Efficient mucosal vaccination mediated by the neonatal Fc receptor

ABSTRACT

The present invention relates to methods and compositions for enhancing delivery of vaccine antigens to the mucosal epithelium, the composition comprising an antigen from an infectious agent fused with an Fc fragment of an immunoglobulin recognized by the neonatal receptors (FcRn). The composition is effective in eliciting a protective long-term memory T cell immune response against infection at a distant mucosal site.

This invention was funded by the National Institutes of Health. TheGovernment has certain rights in the invention pursuant to NIH grantR01AI065892, AI067965, and R21AI073139.

INTRODUCTION

Most pathogens initiate their infections through mucosal surfaces of therespiratory, gastrointestinal and urogenital tracts. An effectivevaccine must therefore induce both mucosal and systemic immune responsesto cope with early infection and pathogen spread (Neutra, M. R. &Kozlowski, P. A., 2006, Nat. Reb. Immunol 6, 148-158; Holmgren, J. &Czerkinsky, C., 2005, Nat. Med. 11 Suppl S45-S53; McGhee, J. R. et al.,1992, Vaccine 10, 75-88; Gallichan, W. S. & Rosenthal, K. L., 1998, J.Infect. Dis. 177, 1155-1161). Delivery of vaccine antigens through themucosal surface would be an ideal route to achieve mucosal, andpotentially, systemic immunity because of the close association betweenmucosal epithelial cells and the immune effector cells within the laminapropria (Neutra and Kozlowski, 2006, supra; Holmgren and Czerkinsky,2005, supra; McGhee et al., 1992, supra). However, since epithelialmonolayers lining the mucosal surfaces are impervious to macromoleculediffusion due to their intercellular tight junctions (Neutra, M. R. etal., 2001, Nat. Immunol. 2, 1004-1009), the mucosal epithelium is anatural barrier for vaccine delivery. Different approaches have beenexplored to circumvent this problem, such as targeting mucosal vaccinesonto differentiated microfold (M) cells that punctuate the mucosalepithelium (Nochi, T. et al. 2007, J. Exp. Med. 204, 2789-2796).However, since columnar epithelial cells comprise the great majority ofmucosal surfaces, alternative mucosal vaccine delivery strategies thattarget these abundant epithelial cells may increase the efficacy ofmucosal vaccines.

Therefore, there is a need for a mucosal vaccine able to penetrate themucosal epithelia and induce a systemic immune response that is bothprotective and long-lasting.

BRIEF DESCRIPTION OF THE INVENTION

The present invention satisfies the need above. In this application isdescribed a method for mucosal vaccination which is shown to provideprolonged protection from infection at a site different than thevaccination site.

The present inventors took advantage of the ability of neonatal Fcreceptor (FcRn) to transport immunoglobulin G (IgG) antibody acrossmucosal surfaces. FcRn is a MHC class I-related molecule which allowsfetuses or newborns to obtain maternal IgG via the placental orintestinal route (Ghetie, V. and Ward, E. S., 2000, Annu. Rev. Immunol.18, 739-766; He, W. et al., 2008, Nature 455, 542-546). FcRn is known toalso transport IgG antibody across mucosal surfaces in adult life(Dickinson, B. L. et al., 1999, J. Clin. Invest. 104, 903-911;Roopenian, d. C. and Akilesh, S., 2007, Nat. Rev. Immunol. 7, 715-725;Baker, K. et al., 2009, Semin. Immunopathol. 31, 223-236; Yoshida, M. etal., 2006, J. clin. Invest. 116, 2142-2151) and lead to resistance tointestinal pathogens (Yoshida et al., 2006, supra). The FcRn receptorbinds IgG (but not other immunoglobulin classes such as IgA, IgD, IgMand IgE) at a relatively lower pH, actively transports the IgGtranscellularly in a luminal to serosal direction or vice versa, andthen releases the IgG at a relatively higher pH found in theinterstitial fluids or luminal surfaces. Observations of IgG transportacross mucosal epithelia by FcRn imply that FcRn may also transport anantigen, if fused with the IgG Fc-fragment (Fc), across the mucosalbarrier. Therefore, FcRn-mediated mucosal vaccine delivery, if feasible,may allow the host to specifically sample an Fc-fused subunit vaccine inthe mucosal lumen, followed by transport of an intact antigen across themucosal epithelial barrier.

Using two different model antigens from two different pathogens whichcause disease and initiate infection at the mucosa of the genital tractof the subject, and further defined the protective immune response. Thepresent inventors determined the ability of FcRn to deliver a vaccine,comprising a fusion of an Fc-fragment from an immunoglobulin subclasswhich binds the FcRn in the mucosa of the subject, with an antigen froma pathogen of interest, across the respiratory mucosal barrier.Intranasal administration of a gD antigen from herpes simplex virustype-2 (HSV-2), or the p24 protein from HIV Gag, fused to theFc-fragment administered along with a CpG adjuvant resulted in local andsystemic immunity including durable memory responses by B and T cells.The immune response was sufficient for protection against intravaginalchallenge with the pathogen source of the antigen.

Immunological memory, an important criterion for any vaccine, is theformation and maintenance of a reservoir of memory T and B lymphocyteswith both adequate size and quality to maintain efficient immunesurveillance for prolonged periods thereby providing the host withability to respond faster and more vigorously to a second encounter withthe pathogen or vaccine antigen (Ahmed, R. and Gray, D., 1996, Science272, 54-60; Bernasconi, N. L. et al., 2002, Science 298, 2199-2202). Todate, immunological memory (Bernasconi, et al., 2002) has been a concernin protein-based subunit mucosal vaccine development becausepreparations elicited levels of immunity immediately after vaccinationbut that immunity waned rapidly over time. The Fc-antigen fusion proteinvaccine described in this application overcomes this drawback.

A striking feature in this study is that FcRn targeted mucosalimmunization promoted and sustained high levels of antigen-specificplasma cells and memory B and T cells at least 6 months after the boostconfirming that the Fc-antigen fusion proteins induce strong antibodyand cellular immune responses at both mucosal and systemic sites againstthe vaccine antigen.

Therefore, the present invention provides a composition and method fordelivering a desired antigen across the mucosal epithelium. The methodcomprises taking advantage of the ability of the neonatal Fc receptor(FcRn) to transport IgG molecules through epithelial cells present inthe respiratory, intestinal and other mucosal epithelia. The compositioncomprises the desired antigen fused to an Fc-fragment of animmunoglobulin recognized by the FcRn of the subject. When this fusionmolecule is introduced to a mucosal tissue, the FcRn mediates thetransport of the Fc-antigen fusion across polarized epithelial cellslining mucosal surfaces. When the antigen is from an infectious agent,transport of the Fc-antigen through the mucosal surface results ininduction of both a humoral and systemic immune response protectiveagainst challenge with the infectious agent at a site different than thevaccination site.

Therefore, it is an object of the present invention to provide a methodfor transporting a desired antigen across a mucosal epithelium,comprising fusing said antigen with a Fc-fragment of an IgG to producean Fc-antigen fusion protein and introducing said Fc-antigen fusionprotein to a mucosal epithelium.

It is another object of the present invention to provide a compositioncomprising a nucleic acid encoding a Fc-fragment from an IgG for use inproducing a fusion protein with the desired vaccine antigen. In oneembodiment, the Fc encoded by the nucleic acid is comprised of the hingeregion, and the CH2 and CH3 domains. Vectors comprising said nucleicacid and host cells transformed with said vectors are also an object ofthe invention.

It is yet another object of the present invention to provide novelvector constructs for cloning a desired antigen such that an Fc-antigenfusion protein can be recombinantly expressed, as well as host cellstransformed with said vector.

It is also another object of the present invention to providecompositions comprising a fusion protein comprising a Fc-fragment of anIgG and a desired antigen.

It is yet another object of the present invention to provide a methodfor producing and purifying a fusion protein comprising a Fc-fragmentfrom an IgG and a desired antigen, comprising:

growing a host cell containing a vector capable of expressing anFc-antigen fusion protein in a suitable culture medium,

causing expression of said vector sequence as defined above undersuitable conditions for production of soluble protein and,

and recovering said Fc-antigen fusion protein.

It is also an object of the present invention to provide kits for thepreparation of an Fc-antigen fusion protein.

It is yet another object of the present invention to provide a mucosalvaccine, with or without adjuvant, comprising a Fc-antigen fusionprotein of the present invention, wherein the antigen is from aninfectious agent, in an amount effective to elicit an immune response inan animal against said infectious agent; and a pharmaceuticallyacceptable diluent, carrier, or excipient. The vaccine according to thepresent invention is inherently safe, is not painful to administer, andshould not result in adverse side effects to the vaccinated individual.

It is another object of the present invention to provide a mucosal DNAvaccine comprising a polynucleotide or hybrid DNA encoding an Fc-antigenfusion peptide.

It is another object of the present invention to provide a method foreliciting in a subject an immune response against an infectious agent,the method comprising administering to a subject a DNA fragmentcomprising a Fc-antigen hybrid DNA.

It is another object of the present invention to provide a method foreliciting in a subject a mucosal and/or systemic immune response againstan infectious agent, the method comprising administering to a subject acomposition comprising Fc-antigen fusion protein of the presentinvention.

All the objects of the present invention are considered to have been metby the embodiments as set out below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Design and characterization of HSV-2 gD fused to IgG Fcfragment. (A). Schematic illustration for the genetic fusion of HSV-2 gDand murine Fcg2a cDNA to create a gD-Fc fusion gene. Mutations were madein the CH2 domain of Fcg2a fragment by using site-directed mutagenesisto replace Glu318, Lys320, and Lys322 with Ala residues to remove thecomplement C1q binding site, and His 310 and His 433 with Ala residuesto eliminate FcRn binding sites. The fourteen codons for glycine andserine residues (GSGGGGSGGGGSGS, SEQ ID NO:1) were inserted between thegD and Fc fragments.

(B). The gD-Fc fusion proteins were secreted by CHO cells. The gD-Fc wasrecognized by either rabbit anti-mouse IgG (top panel) or a mAb anti-gD(bottom panel). The fusion protein was exhibited as a dimer undernon-reducing (NR) or a monomer under reducing (R) condition.

FIG. 2. FcRn-targeted mucosal vaccination induces enhanced gD-specificantibody and T cell responses. The 20 ug gD-Fc/wt, gD-Fc/mut, gD, or PBSin combination with 20 ug CpG were i.n. administered into wild-type (WT)or FcRn knockout (KO) mice.

(A). Measurement of anti-HSV-2 gD-specific IgG antibody titers in serumbefore and after the boost immunization. HSV-2 gD-specific IgG antibodyat indicated days was measured in serum by ELISA. Immunizationconditions are displayed at the right. The curves represent mean valuesfor each group (±S.E.M.). Values marked with asterisk in this andsubsequent figures: *P<0.05; **P<0.01.(B). Test of neutralizing activity in the immunized sera. Sera wereheat-inactivated, diluted 10-fold, then in twofold steps in MEM with 2%FBS. HSV-2 (50 PFU) was added and incubated at 37° C. for 1 hr. Finally,the mixture were removed and washed, overlaid with 0.8% methylcellulosein 2% FBS containing DMEM and further incubated for 72 hr at 37° C. Thetiters were expressed as the reciprocal of the twofold serial dilutionpreventing the appearance of the cytopathic effects (CPE) over controlsera. Each assay was done in triplicate.(C). The percentage of IFN-γ producing T cells in the spleen 4 daysafter the boost. Spleen cells from the immunized mice were stimulatedfor 10 hr with purified gD or medium control. Lymphocytes were gated byforward and side scatter and T cells labeled with anti-CD3 andidentified by their respective surface markers CD4 and CD8 andintracellular IFN-g staining. Immunization conditions are displayed onthe bottom. Numbers represent the percentage of IFN-g⁺ CD3⁺ CD4⁺ (leftpanel) or IFN-g⁺ CD3⁺ CD8⁺ (right panel) T cells. Isotype controlsincluded FITC-mouse-IgG1 with baseline response.(D) Cytokine secretions from the stimulated spleen T cells. Spleen cellswere collected on day 4 after the boost. Cells were stimulated in vitrospecifically with different multiplicity of infection (MOI) ofinactivated HSV-2 virus as indicated for 24 hr. Cytokines IFN-g, IL-2,and IL-4 in the culture supernatant were detected by ELISA. Data arerepresentative of three experiments with three immunized mice pooled ineach group.

FIG. 3. Local immune responses induced by FcRn-targeted mucosalimmunization.

(A). Accumulation of activated B cells in germinal centers (GCs) in themediastinal lymph nodes (MeLNs) and spleen. Representative flowcytometric analyses of GC B cells among CD19+B220+ B cells in the MeLNsand spleen 10 days after the boost. B220⁺PNA^(high) cells are B cellsthat exhibit the phenotypic attributes of GC B cells. The GC staining inspleen was used as a positive control. Numbers are the percentage ofactivated GC B cells (PNA+FAS+) among gated B cells and arerepresentative of three independent experiments.(B). GC formation and presence of activated B cells followingimmunizations as indicated. Frozen MeLN sections at day 10 from theimmunized mice were co-stained with biotin-PNA (developed withavidin-FITC) and Alexa647 labeled anti-IgD. Scale bar represents 50 mm.(C). Quantitative analysis of GCs following immunization. The dynamicsof the frequency of germinal center B cells (FAS+PNA+, gated onCD19+B220+ cells) were plotted on day 10, 22 and 35 after the boost.Data indicate the mean and S.E.M., n=5 mice.(D). The formation of inducible bronchus-associated lymphoid tissue(iBALT). Frozen serial sections of the lung were stained with biotin-PNA(GC, red) and anti-B220 (B cells, green), followed by Alexa488-conjugated IgG of corresponding species and Alexa 555-Avidin. Thenucleus is stained with DAPI (blue). A germinal center-like structure isshown in the merged panel by the white color. The data arerepresentative of sections from at least three independent mice. Imageswere originally obtained at 10× magnification. Scale bars represent 100μm.(E)+(F). Presence of HSV-2 gD-specific T lymphocytes in the lung (E) andMeLNs (F). Lung or MeLN cells from mice 4 days after the boost werecollected. Lymphocytes were gated based on their forward scatter (FSC)vs. side scatter (SSC) profile. Intracellular staining for IFN-g, wasperformed after surface staining of CD4 and CD8 molecules. The profilesshown are representative of five mice from three separate experiments.Numbers indicate percentages of IFN-g-producing T lymphocytes from gatedCD4⁺ and CD8⁺ T cells.

FIG. 4. FeRn-targeted mucosal immunization provides protective immunityto intravaginal (ivag) challenge with virulent HSV-2 186.

(A) Mean survival following genital HSV-2 challenge. Four weeks afterthe immunization, groups of five mice were challenged intravaginallywith 1×10⁴ pfu of HSV-2 strain 186. Percentage of mice from protectionon the indicated days is calculated as the number of mice survivingdivided by the number of mice in each group and represented two similarexperiments.(B) Mean of viral titers following HSV-2 challenge. Virus titers weremeasured from vaginal washes by taking swabs on the indicated days afterHSV-2 inoculation based on a plaque assay on Vero cell monolayers.(C). Increased presence of HSV-specific T lymphocytes in the vaginalepithelium after challenge. Lymphocytes were harvested fromcollagenase-digested vaginal tissues 4 days intravaginal inoculation ofvirus. Intracellular staining for IFN-g expression on CD4⁺ and CD8⁺ Tcells was analyzed after gating on viable CD3+ lymphocytes. The numbersin each column show the percentage of IFN-g-positive T lymphocytes fromthe gated CD4⁺ or CD8⁺ T cells. Data shown are of a representative fromthree experiments using 3 mice per experiment.

FIG. 5. Increased memory immune response in FcRn-targeted mucosalimmunization.

(A). Induction of gD specific memory B cells in the spleen. Thefrequency of gD-specific memory B cells was assessed 6 months after theboost. Memory B cells, defined as B220⁺ gD-surface⁺, were analyzed 6months after the boost by FACS. Purified gD proteins were labeled withAlexa Fluoro647. Spleen cells (2×10⁶) were incubated with the 1 mg AlexaFluoro647-labeled gD proteins and B220 antibody. Numbers in thequadrants are the percentage of gD-specific memory B lymphocytes.(B). Long-lived HSV gD-specific antibody-secreting cells in the bonemarrow. Bone marrow cells removed 6 months after the boost were placedon gD-coated plates and quantified by ELISPOT analysis of IgG-secretingplasma cells. Data were pooled from two separate experiments with fivemice in each experiment. The graphs were plotted based on the averageELISPOT for replicate wells. Values marked with asterisk aresignificantly greater (P<0.01) from the gD-Fc/wt protein-immunized micethan those of other groups as indicated.(C). Durability of HSV-2 gD-specific serum IgG response. In two separateexperiments, HSV-2 gD-specific IgG was quantified by ELISA in serum byendpoint titer from five mice at 6 months after the boost. HSV-specificIgG antibody was not detected in PBS-immunized mice.(D). Long-lived gD specific T cell memory to FcRn-targeted mucosalvaccination. Spleen cells were isolated from the immunized mice sixmonths after the boost, stained with CFSE, and stimulated in vitro with20 mg/ml of purified gD for 4 days. Data are expressed in CFSEhistograms of fluorescence intensity versus the number of fluorescingcells, indicating the percentage of the cell population positive for CD4and CD8 antigen. Numbers in the quadrants are the percentage of CD4⁺ andCD8⁺ proliferating T cells. Representative flow cytometry profiles oftwo similar experiments with three mice per group are shown.Immunization conditions are displayed on the top.(E). Mean survival following genital HSV-2 challenge six monthsfollowing the boost. The immunized mice were challenged intravaginallywith 1×10⁴ pfu of HSV-2. Percentage of mice protected on the indicateddays is calculated as the number of mice surviving divided by the numberof mice in each group (n=5).(F). Proposed model of FcRn-mediated mucosal vaccine delivery. TheFc-fused antigens are transported by FcRn and targeted to the mucosalantigen presenting cells (APCs), such as dendritic cells. Antigen istaken up by pinocytosis or FcgRI-mediated endocytosis in APCs, thenprocessed and presented to T cells.

FIG. 6. Design and characterization of HIV-1 Gag fused to IgG Fcfragment and FcRn-dependent transcytosis of the Gag-Fc/wt.

(A). Schematic illustration for the genetic fusion of HIV Gag and murineFcγ2a cDNA to create a Gag-Fc fusion gene. Mutations were made in theCH2 domain of Fcg2a fragment by using site-directed mutagenesis toreplace Glu318, Lys320, and Lys322 with Ala residues to remove thecomplement C1q binding site, and His 310 and His 433 with Ala residuesto eliminate FcRn binding sites.(B). The Gag-Fc fusion proteins were secreted by CHO cells. The Gag-Fcwas recognized by either a mAb anti-Gag (top panel) or rabbit anti-mouseIgG (bottom panel). The fusion protein appeared as a dimer undernon-reducing (NR) or a monomer under reducing (R) condition.(C). Transport of Gag-Fc/wt fusion proteins in MDCK-FcRn cell lines.MDCK-FcRn cells were plated onto 24-mm transwells and grown for 3-6 daysto allow the formation of a polarized monolayer with resistance greaterthan 300 Ωcm². Purified Gag-Fc/wt (100 mg/ml) and chicken IgY wereapplied to the apical reservoir and transcytosis was allowed to proceedfor 2 hr. The proteins were collected from the basolateral reservoir andblotted with anti-Gag antibody under reducing condition. The Gag-Fc/wtfusion protein (lanes 4&5, top panel), but not Gag-Fc/mut (lanes 2&3,top panel) or IgY (bottom panel), was detected by Western blot. Lane 1,representing Gag-Fc/wt or IgY protein, was used as a positive control.wt: wild-type; mut: mutant.(D). Expression of mouse FcRn in the trachea and lung in adult mice.Frozen sections of tissue samples obtained from wt or FcRn KO mice werestained with affinity purified rabbit anti-FcRn antibody and followed byAlexa Fluoro 488-conjugated IgG (green). FcRn staining was not observedin the presence of normal rabbit IgG. The nucleus is stained with DAPI(blue). The data are representative of sections from at least threeindependent mice. Images were originally obtained at 40× magnification.Scale bars represent 20 μm.(E). Transport of the Gag-Fc/wt proteins across mucosal barrier.Purified, Gag, Gag-Fc/wt and Gag-Fc/mut proteins (20 mg) wereintranasally inoculated into wild-type and FcRn KO mice as indicated. 8hr later, the mouse sera were collected, the Gag or Gag-Fc proteinconcentration in blood circulation was measured by ELISA. Inoculationconditions are displayed. Star (**) denotes p<0.01.(F). Persistence of the Gag-Fc/proteins in the sera. The proteinconcentrations in sera were measured by ELISA after the transfer atindicated time (hr).

FIG. 7. FcRn-targeted mucosal vaccination induces enhanced Gag-specificantibody and T cell immune responses. 20 ug Gag-Fc/wt, Gag-Fc/mut, Gagalone with 20 ug CpG were i.n. administered into wild-type or FcRn KOmice.

(A). Measurement of anti-HIV Gag-specific IgG antibody titers in serumbefore and after the boost immunization. HIV Gag-specific IgG antibodyat indicated days was measured in serum by ELISA. Immunizationconditions are displayed at the right.

(B). Measurement of HIV Gag-specific IgG isotype titers in the serum.Blood samples were taken from mice by tail bleeding. HIV Gag-specificIgG isotype at 28 days was measured in serum by ELISA. Immunizationconditions are displayed in the bottom.

(C). Detection of activated B cells in the germinal center (GC) in theimmunized mice by flow cytometry. Representative flow cytometricanalyses of GC B cells among B220⁺ B cells in the spleen 10 days afterthe boost. Numbers are the percentage of activated GC B cells (PNA⁺FAS⁺)among gated B220⁺ cells.(D). The percentage of IFN-γ producing T cells in the spleen 7 daysafter the boost. Splenocytes from the immunized mice were stimulated for18 hr with purified Gag or medium control. Lymphocytes were gated byforward and side scatter and T cells labeled with anti-CD3 andidentified by their respective surface markers CD4 and CD8 andintracellular IFN-g staining. Immunization conditions are displayed onthe top. Numbers in the quadrants represent the percentage of IFN-γ⁺CD3⁺ CD4⁺ (top panel) or IFN-γ⁺ CD3⁺ CD8⁺ (bottom panel) T cells.Isotype controls included FITC-mouse-IgG1 and show baseline response.(E). Cytokine secretions from the stimulated spleen T cells. Splenocyteswere collected and pooled from three immunized mice per group on day 7after the boost. Cells were stimulated in vitro specifically withpurified Gag for 24 hr. Cytokines IFN-g, IL-2, and IL-4 in the culturesupernatant were detected by ELISA. They are presented as picograms/mlof culture supernatant. Data are representative of three experimentswith three mice pooled in each experiment.

FIG. 8. FcRn-targeted mucosal immunization engenders protective immunityto intravaginal viral challenge.

(A). Mean of viral titers in ovaries following intravaginal challengewith vaccinia virus (VV) expressing HIV-1 Gag (rVV-Gag). Four weeksafter the boost, groups of five mice were intravaginally challenged with5×10⁷ pfu of rVV-Gag. Mice were sacrificed 5 days after infection andpaired ovaries were collected. Ovaries were homogenized and viral titerswere determined by standard plaque assay on Vero cell monolayer. Thedata represented three similar experiments.(B). Macroscopic pictures of uteri from normal mice or the immunizedmice challenged with rVV-Gag are shown. Immunization conditions aredisplayed in the bottom.

FIG. 9. Local immune responses induced by FcRn-targeted mucosalimmunization.

(A). Detection of activated B cells in the germinal center (GC) in theimmunized mice by flow cytometry. Representative flow cytometricanalyses of GC B cells among B220⁺ B cells in the mediastinal lymph node(MLN) 10 days after the immunization. Numbers are the percentage ofactivated GC B cells (PNA⁺FAS⁺) among gated B220⁺ cells.(B+C). HIV Gag-specific antibody responses in bronchial alveolar lavage(BAL) and vaginal secretions following immunization. BAL (B) and vaginalwashes (C) were obtained from mice 10 days after the boost andGag-specific IgG titers were determined by ELISA. Antibody titers for 3mice from a representative experiment were quantified by endpoint titer.Titers of HIV Gag-specific IgG antibody from BAL and vaginal washes ofnaive mice always fell below the limit of detection and are omitted fromthe figure for clarity. The data shown are representative of threeindependent experiments. Asterisk (*) indicates significant differenceamong groups (P≦0.05).(D). Increased presence of HIV Gag-specific T lymphocytes in the vaginalepithelia after challenge. Lymphocytes were harvested fromcollagenase-treated vaginal tissues 5 days after intravaginalinoculation of rVV-Gag. Intracellular staining for IFN-g expression onCD4+ and CD8+ T cells was analyzed after gating on viable CD3+lymphocytes. The numbers in each column show the percentage ofIFN-g-positive T lymphocytes from the gated CD4⁺ or CD8⁺ T cells.Isotype controls included FITC conjugated mouse-IgG1 and show baselineresponse. Data shown are of a representative from three experimentsusing 3 mice per experiment.

FIG. 10. Increased memory immune response in FcRn-targeted mucosalimmunization.

(A). Induction of Gag specific memory B cells in the spleen. Thefrequency of Gag-specific memory B cells was assessed 4 months after theboost. The Gag-specific memory B cells, defined as B220⁺IgG⁺IgD⁻, wereanalyzed 4 months after the boost by FACS. Purified HIV Gag proteinswere labeled with Alexa Fluoro647. Splenocytes (2×10⁶) were incubatedwith the 1 ug Alexa Fluoro647-labeled Gag proteins and B220 antibody.Numbers in the quadrants are the percentage of HIV Gag-specific memory Blymphocytes.(B). Long-lived HIV Gag-specific antibody-secreting cells in the bonemarrow. Bone marrow cells removed 4 months after the boost were placedon Gag-coated plates and quantified by ELISPOT analysis of IgG-secretingplasma cells. Data were pooled from three separate experiments withthree mice in each experiment. The graphs were plotted based on theaverage ELISPOTs for replicate wells (top panel). Values marked withasterisk are significantly greater (P<0.01) from the Gag-Fc/wt fusionprotein-immunized mice than those for other groups as indicated in thebottom.(C). Durability of HIV Gag-specific serum IgG response. In two separateexperiments, HIV Gag-specific IgG was quantified by ELISA in serum byendpoint titer from three mice at 4 months after the boost. HIV-specificIgG antibody was not detected in naive mice.D). Long-lived HIV Gag-specific T cell memory response to FcRn-targetedmucosal vaccination. Splenocytes were isolated four months after theboost, stained with CFSE, and stimulated in vitro with 20 ug/ml ofpurified Gag for 4 days. Data are expressed in CFSE histograms offluorescence intensity versus the number of fluorescing cells,indicating the percentage of the cell population positive for CD4 or CD8antigen. Numbers in the quadrants are the percentage of CD4⁺ and CD8⁺proliferating T cells. Data are representative flow cytometry profilesof two similar experiments with three mice per group. Immunizationconditions are displayed on the top.(E). Mean of viral titers in ovaries following vaginal challenge withrVV-Gag. Four months after the boost, groups of five mice wereintravaginally challenged with 5×10⁷ pfu of rVV-Gag and sacrificed 5days after challenge. Ovaries were collected for each mouse and viraltiters were measured by a plaque assay. The data represented threesimilar experiments.(F). Proposed model of FcRn-mediated mucosal vaccine delivery. TheFc-fused HIV gag antigens are transported by FcRn across epithelialmucosal barrier and targeted to the mucosal antigen presenting cells(APCs), such as dendritic cells. Antigen is taken up by pinocytosis orFcgRI-mediated endocytosis in APCs, then processed and presented orcross presented to T cells. TCR: T cell receptor; APC, antigenpresenting cells.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in recombinantDNA and immunology are extensively utilized. In order to provide aclearer and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

The term ‘biological sample’ intends a fluid or tissue of a mammalianindividual (e.g. an anthropoid, a human), reptilian, avian, or any otherzoo or farm animal that commonly contains antibodies produced by theindividual. Such components are known in the art and include, withoutlimitation, blood, plasma, serum, urine, spinal fluid, lymph fluid,secretions of the respiratory, intestinal or genitourinary tracts,tears, saliva, milk, white blood cells and myelomas. Body componentsinclude biological liquids. The term ‘biological fluid’ refers to afluid obtained from an organism. Some biological fluids are used as asource of other products, such as clotting factors (e.g. Factor VIII),serum albumin, growth hormone and the like.

The term ‘immunologically reactive’ means that the antigen in questionwill react specifically with antibodies present in a body component froman infected individual.

The term ‘immune complex’ intends the combination formed when anantibody binds to an antigen.

The term ‘purified’ as applied to proteins herein refers to acomposition wherein the desired protein comprises at least 35% of thetotal protein component in the composition. The desired proteinpreferably comprises at least 40%, more preferably at least about 50%,more preferably at least about 60%, still more preferably at least about70%, even more preferably at least about 80%, even more preferably atleast about 90%, and most preferably at least about 95% of the totalprotein component. The composition may contain other compounds such ascarbohydrates, salts, lipids, solvents, and the like, without affectingthe determination of the percentage purity as used herein. An ‘isolated’Fc-antigen fusion protein intends a fusion protein composition that isat least 35% pure.

The term ‘recombinantly expressed’ used within the context of thepresent invention refers to the fact that the proteins of the presentinvention are produced by recombinant expression methods be it inprokaryotes, or lower or higher eukaryotes as discussed in detail below.

The term ‘lower eukaryote’ refers to host cells such as yeast, fungi andthe like. Lower eukaryotes are generally (but not necessarily)unicellular.

Preferred lower eukaryotes are yeasts, particularly species withinSaccharomyces. Schizosaccharomyces, Kluveromyces, Pichia (e.g. Pichiapastoris), Hansenula (e.g. Hansenula polymorpha, Yarowia, Schwaniomyces,Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomycescerevisiae, S. carlsberoensis and K. lactis are the most commonly usedyeast hosts, and are convenient fungal hosts.

The term ‘prokaryotes’ refers to hosts such as E. coli, Lactobacillus,Lactococcus, Salmonella, Streptococcus, Bacillus subtilis orStreptomyces. Also these hosts are contemplated within the presentinvention.

The term ‘higher eukaryote’ refers to host cells derived from higheranimals, such as mammals, reptiles, insects, and the like. Presentlypreferred higher eukaryote host cells are derived from Chinese hamster(e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK),pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcomacell line 143 B, the human cell line HeLa and human hepatoma cell lineslike Hep G2, and insect cell lines (e.g. Spodoptera frugiperda). Thehost cells may be provided in suspension or flask cultures, tissuecultures, organ cultures and the like. Alternatively the host cells mayalso be transgenic animals.

The term ‘polypeptide’ refers to a polymer of amino acids and does notrefer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like.

Included within the definition are, for example, polypeptides containingone or more analogues of an amino acid (including, for example,unnatural amino acids, PNA, etc.), polypeptides with substitutedlinkages, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring.

The term ‘recombinant polynucleotide or nucleic acid’ intends apolynucleotide or nucleic acid of genomic, cDNA, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation: (1) isnot associated with all or a portion of a polynucleotide with which itis associated in nature, (2) is linked to a polynucleotide other thanthat to which it is linked in nature, or (3) does not occur in nature.

The term ‘recombinant host cells’, ‘host cells’, ‘cells’, ‘cell lines’,‘cell cultures’, and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be or have been, used as recipients for a recombinant vectoror other transfer polynucleotide, and include the progeny of theoriginal cell which has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

The term ‘replicon’ is any genetic element, e.g., a plasmid, achromosome, a virus, a cosmid, etc., that behaves as an autonomous unitof polynucleotide replication within a cell; i.e., capable ofreplication under its own control.

The term ‘vector’ is a replicon further comprising sequences providingreplication and/or expression of a desired open reading frame.

The term ‘control sequence’ refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and terminators; in eukaryotes,generally, such control sequences include promoters, terminators and, insome instances, enhancers. The term ‘control sequences’ is intended toinclude, at a minimum, all components whose presence is necessary forexpression, and may also include additional components whose presence isadvantageous, for example, leader sequences which govern secretion.

The term ‘promoter’ is a nucleotide sequence which is comprised ofconsensus sequences which allow the binding of RNA polymerase to the DNAtemplate in a manner such that mRNA production initiates at the normaltranscription initiation site for the adjacent structural gene.

The expression ‘operably linked’ refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence ‘operably linked’to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

An ‘open reading frame’ (ORF) is a region of a polynucleotide sequencewhich encodes a polypeptide and does not contain stop codons; thisregion may represent a portion of a coding sequence or a total codingsequence.

A ‘coding sequence’ is a polynucleotide sequence which is transcribedinto mRNA and/or translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include but is not limited to mRNA, DNA (including cDNA),and recombinant polynucleotide sequences.

The term ‘immunogenic’ refers to the ability of a substance to cause ahumoral and/or cellular response, whether alone or when linked to acarrier, in the presence or absence of an adjuvant. ‘Neutralization’refers to an immune response that blocks the infectivity, eitherpartially or fully, of an infectious agent. A ‘vaccine’ is animmunogenic composition capable of eliciting protection against disease,whether partial or complete. A vaccine may also be useful for treatmentof an infected individual, in which case it is called a therapeuticvaccine.

The term ‘therapeutic’ refers to a composition capable of treating adisease or infection. The term ‘effective amount’ for a therapeutic orprophylactic treatment refers to an amount of epitope-bearingpolypeptide sufficient to induce an immunogenic response in theindividual to which it is administered, or to otherwise detectablyimmunoreact in its intended system (e.g., immunoassay). Preferably, theeffective amount is sufficient to effect treatment, as defined above.The exact amount necessary will vary according to the application. Forvaccine applications or for the generation of polyclonalantiserum/antibodies, for example, the effective amount may varydepending on the species, age, and general condition of the individual,the severity of the condition being treated, the particular polypeptideselected and its mode of administration, etc. It is also believed thateffective amounts will be found within a relatively large, non-criticalrange. An appropriate effective amount can be readily determined usingonly routine experimentation. Preferred ranges of for prophylaxis ofdisease are about 0.01 to 1000 ug/dose, more preferably about 0.1 to 100ug/dose, most preferably about 10-50 ug/dose. Several doses may beneeded per individual in order to achieve a sufficient immune responseand subsequent protection against disease.

More particularly, the enhanced delivery methods and compositions of thepresent invention provide for effective mucosal delivery of anFc-antigen vaccine for prevention or treatment of disease or infectionin mammalian subjects.

The invention is useful whenever it is desirable to deliver an antigenacross an epithelial barrier to the immune system. The invention thusmay be used to deliver antigens across intestinal epithelial tissue,lung epithelial tissue and other mucosal surfaces including nasalsurfaces, vaginal surfaces, and colon surfaces. The invention may beused to induce in a subject an immune response by stimulating a humoralantibody response against an antigen, or by stimulating T cell activity.As used herein, subject means: humans, primates, horses, cows, sheep,pigs, goats, dogs, cats, chickens and rodents.

The invention involves the formation of a fusion protein comprising theFc-fragment of an IgG and an antigen. A fusion protein is a polypeptideresulting from the expression of a hybrid DNA, the hybrid DNA created byfusing two or more nucleic acids encoding two or more proteins orpeptides.

The region of the Fc-fragment of IgG that binds to the FcRn receptor inhumans has been described based upon X-ray crystallography (Burmaister,W. P. et al., Nature, 1994; 372:379-378.) The major contact area of Fcwith the FcRn receptor is near the junction of the C_(H2) and C_(H3)domains. Potential contacts are residues 248, 250-257, 272, 285, 288,290-291, 308-311 and 314 in C_(H2) and 385-387, 428 and 433-436 inC_(H3). The foregoing Fc-FcRn contacts are all within a single Ig heavychain. Within the scope of the invention are nucleotide sequencesencoding human Fc.

It has been noted previously that two FcRn receptors can bind a singleFc molecule. The crystallographic data suggest that in such a complex,each FcRn molecule binds a single polypeptide of the Fc homodimer.Therefore, in another aspect, a Fc heterodimer is provided wherein eacharm of the Fc molecule is fused to a different antigen, whether theantigens are for the same disease or for different diseases.Alternatively, one Fc arm is fused to a desired antigen and the other Fcarm is fused to an adjuvant of interest. It is understood thatvariations on antigens, such as, among others, chimeric antigensproduced from fusing two or more immunogenic epitopes from two or moreantigens into one fusion protein, then fused to Fc, are within the skillof a person in the art.

The Fc-fragment should be chosen from an immunoglobulin known to bindthe FcRn in the mucosa of the subject receiving the antigen-Fc vaccine.Immunoglobulin subclasses recognized by FcRn in different epithelialmucosa of animal subjects are known to a person in the art and can befound in Ober, R. J. et al, 2001, Int. Immunol. 13, 1551-9.

In accordance with the present invention, the Fc-antigen fusion proteinmay be produced by recombinant genetic engineering techniques known inthe art, for example, DNA ligation, or PCR-based gene assembly asdescribed in the Examples below. Other methods known in the art can beused.

Given the foregoing information, those of ordinary skill in the art willreadily recognize that the Fc region of IgG can be modified according towell-recognized procedures such as site-directed mutagenesis and thelike to yield modified IgG or modified Fc fragments or portions thereofthat will be bound by the FcRn receptor. Such modifications includemodifications remote from the FcRn contact sites as well asmodifications within the contact sites that preserve or even enhancebinding. If the Fc-antigen fusion protein is composed entirely ofgene-encoded amino acids, or a portion of it is so composed, the fusionprotein or the relevant portion may also be synthesized usingconventional recombinant genetic engineering techniques.

For recombinant production, established methods (Sambrook et al.,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 1989) would be used to engineer DNA encoding afusion protein comprised of the antigenic peptide or protein and a Fc.This DNA would be placed in an expression vector and introduced intobacterial or eukaryotic cells by established methods. The fusion proteinwould be purified from the cells or from the culture medium byestablished methods. Methods for recombinant protein and peptideproduction are well known in the art (see, e.g., Maniatis et al., 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.).

In accordance with the present invention, an Fc may be fused to apeptide or protein derivative such as those listed herein including, butnot limited to, antigens, allergens, pathogens or to other proteins orprotein derivatives of potential therapeutic interest such as growthfactors, colony stimulating factors, growth inhibitory factors,signaling molecules, hormones, steroids, neurotransmitters, ormorphogens that would be of use when delivered across an epithelialbarrier.

Included within the definition of biologically active peptides andproteins, or antigens for use within the invention are natural orsynthetic, therapeutically or prophylactically active, peptides(comprised of two or more covalently linked amino acids), proteins,peptide or protein fragments, peptide or protein analogs, and chemicallymodified derivatives or salts of active peptides or proteins.

An antigen as used herein falls into four classes: 1) antigens that arecharacteristic of a pathogen; 2) antigens that are characteristic of anautoimmune disease; 3) antigens that are characteristic of an allergen;and 4) antigens that are characteristic of a tumor. Antigens in generalinclude polysaccharides, glycolipids, glycoproteins, peptides, proteins,carbohydrates and lipids from cell surfaces, cytoplasm, nuclei,mitochondria and the like.

Antigens that are characteristic of pathogens include antigens derivedfrom viruses, bacteria, parasites or fungi. Examples of importantpathogens include vibrio choleras, enterotoxigenic Escherichia coli,rotavirus, Clostridium difficile, Shigella species, Salmonella typhi,parainfluenza virus, influenza virus, Streptococcus pneumonias, Borellaburgdorferi, HIV, Streptococcus mutans, Plasmodium falciparum,Staphylococcus aureus, rabies virus, Epstein-Barr virus, and herpessimplex virus. Specific antigens are known to those of skill in the art,for example, influenza HA, NA, M2, HIV gp120, mycobacterium tuberculosisAg85B and ESAT6, Streptococcus pneumonia PspA, PsaA, and CbpA,respiratory syncytial virus (RSV) F and G protein, human papilloma virusprotein, to name a few.

Viruses in general include but are not limited to those in the followingfamilies: picornaviridae; caliciviridae; togaviridae; flaviviridae;coronaviridae; rhabdoviridae; filoviridae; paramyxoviridae;orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae; retroviridae;hepadnaviridae; parvoviridae; papovaviridae; adenoviridae;herpesviridae; and poxyviridae.

Bacteria in general include but are not limited to: P. aeruginosa; E.coli, Klebsiella sp.; Serratia sp.; Pseudomanas sp.; P. cepacia;Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumonias; S. aureus;Haemophilus sp.; Neisseria Sp.; N. meningitidis; Bacteroides sp.;Citrobacter sp.; Branhamella sp.; Salmonelia sp.; Shigella sp.; S.pyogenes; Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Lesteriasp.; Pasteurella multocida; Streptobacillus sp.; Spirillum sp.;Fusospirocheta sp.; Treponema pallidum; Borrelia sp.; Actinomycetes;Mycoplasma sp.; Chlamydia sp.; Rickettsia sp.; Spirochaeta; Legionellasp.; Mycobacteria sp.; Ureaplasma sp.; Streptomyces sp.; Trichomorassp.; and P. mirabilis.

Parasites include but are not limited to: Plasmodium falciparum, P.vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana, L.tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma mansoni, S. haematobium, S. japonium; Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobius vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocytis carinii, Babesia bovis, B. divergens, B. microti,Isospore belli, L hominis; Dientamoeba fragiles; Onchocerca volvulus;Ascaris lumbricoides, Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongyluscantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westermani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica Sarcoptes scabiei, Pediculus humanus; Phthirius pubis; andDermatobia hominis.

Fungi in general include but are not limited to: Cryptococcusneoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis;Histoplasfria capsulatum; Coccidioides immitis; Candids species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, Aspergillus species, including A. fumigatus, A. flavusand A. niger, Rhizopus species; Rhizomucor species; Cunninghammellaspecies; Apophysomyces species, including A. saksenaea, A. mucor and A.absidia; Sporothrix schenckii, Paracoccidioides brasiliensis;Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.

Antigens that are characteristic of autoimmune disease typically will bederived from the cell surface, cytoplasm, nucleus, mitochondria and thelike of mammalian tissues. Examples include antigens characteristic ofuveitis (e.g. S antigen), diabetes mellitus, multiple sclerosis,systemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, primary myxoedema, thyrotoxicosis, rheumatoid arthritis,pernicious anemia, Addison's disease, scleroderma, autoimmune atrophicgastritis, premature menopause (few cases), male infertility (fewcases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris,pemphigoid, sympathetic opthalmia, phacogenic uveitis, autoimmunehaemolytic anemia, idiopathic thrombocylopenic purpura, idiopathicfeucopenia, pr imary biliary cirrhosis (few cases), ulcerative colitis,Siogren's syndrome, Wegener's granulomatosis, poly/dermatomyositis, anddiscold lupus erythromatosus.

Antigens that are allergens are generally proteins or glycoproteins,although allergens may also be low molecular weight allergenic haptensthat induce allergy after covalently combining with a protein carrier(Remington's Pharmaceutical Sciences). Allergens include antigensderived from pollens, dust, molds, spores, dander, insects and foods.Specific examples include. the urushiols (pentadecylcatechol orheptadecylcatechol) of Toxicodendron species such as poison ivy, poisonoak and poison sumac, and the sesquiterpenoid lactones of ragweed andrelated plants.

Antigens that are characteristic of tumor antigens typically will bederived from the cell surface, cytoplasm, nucleus, organelles and thelike of cells of tumor tissue. Examples include antigens characteristicof tumor proteins, including proteins encoded by mutated oncogenes;viral proteins associated with tumors; and tumor mucins and glycolipids.Tumors include, but are not limited to, those from the following sitesof cancer and types of cancer: lip, nasopharynx, pharynx and oralcavity, esophagus, stomach, colon, rectum, liver, gall bladder, binarytree, pancreas, larynx, lung and bronchus, melanoma of skin, breast,cervix, uteri, uterus, ovary, bladder, kidney, brain and other parts ofthe nervous system, thyroid, prostate, testes, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma and leukemia. Viral proteinsassociated with tumors would be those from the classes of viruses notedabove. Antigens characteristic of tumors may be proteins not usuallyexpressed by a tumor precursor cell, or may be a protein which isnormally expressed in a tumor precursor cell, but having a mutationcharacteristic of a tumor. An antigen characteristic of a tumor may be amutant variant of the normal protein-having an altered activity orsubcellular distribution. Mutations of genes giving rise to tumorantigens, in addition to those specified above, may be in the codingregion, 5′ or 3′ noncoding regions, or introns of a gene, and may be theresult of point mutations frameshifts, deletions, additions,duplications, chromosomal rearrangements and the like. One of ordinaryskill in the art is familiar with the broad variety of alterations tonormal gene structure and expression which gives rise to tumor antigens.Specific examples of tumor antigens include: proteins such asIg-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 ofmelanoma, Pmel-17 (gp 100) of melanoma, MART-1 (Melan-A) of melanoma,p15 protein of melanoma, tyrosinase of melanoma, MAGE 1, 2 and 3 ofmelanoma, thyroid medullary, small cell lung cancer, colon and/orbronchial squamous cell cancer, BAGE of bladder, melanoma, breast, andsquamous-cell carcinoma, gp75 of melanoma, oncofetal antigen ofmelanoma; carbohydrate/lipids such as muci mucin of breast, pancreas,and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes suchas mutant p53 of carcinoma, mutant ras of colon cancer and HER21neuproto-onco-gene of breast carcinoma; viral products such as humanpapilloma virus proteins of squamous cell cancers of cervix andesophagus. It is also contemplated that proteinaceous tumor antigens maybe presented by HLA molecules as specific peptides derived from thewhole protein. Metabolic processing of proteins to yield antigenicpeptides is well known in the art; for example see U.S. Pat. No.5,342,774 (Boon et al.). The present method thus encompasses delivery ofantigenic peptides and such peptides in a larger polypeptide or wholeprotein which give rise to antigenic peptides.

Generally, subjects can receive an effective amount of the tumorantigen, and/or peptide derived therefrom by one or more of the methodsdetailed below. Initial doses can be followed by booster doses,following immunization protocols standard in the art. Delivery of tumorantigens thus may stimulate proliferation of cytolytic T lymphocytes.

In one embodiment, fusion proteins of the present invention areconstructed in which the fusion consists of an Fc fragment (startingwith the amino acids E-P-R-G at the N-terminus of the hinge region,including the CH2 regions, and continuing through the T-P-G-K sequencein CH3 region) fused to one of the above listed proteins.

When administered, the Fc-antigen fusion peptides of the presentinvention are administered in pharmaceutically acceptable preparations.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents.

The Fc-antigen fusion peptides of the invention may be administered in apurified form or in the form of a pharmaceutically acceptable salt. Whenused in medicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmaceutically acceptable saltsinclude, but are not limited to, those prepared from the followingacids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, pharmaceutically acceptable salts can beprepared as alkyline metal or alkyline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2:5,% WN);sodium bicarbonate (0.5-1.0% W/V); and phosphoric acid and a salt(0.8-2% W/V). Suitable preservatives include benzalkonium chloride(0.003-0.03% W/V); chlotubutanol (0.3-0.9% W/V); parabens (0.01-0.25%W/V) and thimerosal (0.004-0.02% W/V).

The term “carrier” as used herein, and described more fully below, meansone or more solid or liquid filler, dilutants or encapsulatingsubstances which are suitable for administration to a human or othermammal. The “carrier” may be an organic or inorganic ingredient, naturalor synthetic, with which the active ingredient is combined to facilitateadministration.

The components of the pharmaceutical compositions are capable of beingcommingled with the Fc-antigen fusion protein of the present invention,and with each other, in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficacy. Thecomponents of oral drug formulations include diluents, binders,lubricants, glidants, disintegrants, coloring agents and flavoringagents.

Encapsulating substances for the preparation of enteric-coated oralformulations include cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate and methacrylic acidester copolymers. Solid oral formulations such as capsules or tabletsare preferred. Elixirs and syrups also are well known oral formulations.The components of aerosol formulations include solubilized activeingredients, antioxidants, solvent blends and propellants for solutionformulations, and micronized and suspended active ingredients,dispersing agents and propellants for suspension formulations. The oral,aerosol and nasal formulations of the invention can be distinguishedfrom injectable preparations of the prior art because such formulationsmay be nonaseptic, whereas injectable preparations must be aseptic.

The term “adjuvant” is intended to include any substance which isincorporated into or administered simultaneously with the fusion proteinof the invention and which nonspecifically potentiates the immuneresponse in the subject. Adjuvants include aluminum compounds, e.g.,gels, aluminum hydroxide and aluminum phosphate, and Freund's completeor incomplete adjuvant (in which the fusion protein is incorporated inthe aqueous phase of a stabilized water in paraffin oil emulsion). Theparaffin oil may be replaced with different types of oils, e.g.,squalene or peanut oil. Other materials with adjuvant propertiesinclude, flagellin, BCG (attenuated Mycobacterium tuberculosis), calciumphosphate, levamisole, isoprinosine, polyanions (e.g., poly A:U)leutinan, pertussis toxin, cholera toxin, lipid A, saponins andpeptides, e.g. muramyl dipeptide. dimethyl dioctadecyl-ammonium bromide(DDA); monophosphoryl lipid A (MPL); LTK63, lipophilic quaternaryammonium salt-DDA, Trehalose dimycolate and synthetic derivatives,DDA-MPL, DDA-TDM, DDA-TDB, IC-31, aluminum salts, aluminum hydroxyide,aluminum phosphate, potassium aluminum phosphate, Montanide ISA-51,ISA-720, microparticles, immunostimulatory complexes, liposomes,virosomes, virus-like particles, CpG oligonucleotides, cholera toxin,heat-labile toxin from E. coli, lipoproteins, dendritic cells, IL-12,GM-CSF, nanoparticles including calcium phosphate nanoparticles,combination of soybean oil, emulsifying agents, and ethanol to form ananoemulsion; AS04, ZADAXIN, or combinations thereof. Rare earth salts,e.g., lanthanum and cerium, may also be used as adjuvants. The amount ofadjuvants depends on the subject and the particular fusion protein usedand can be readily determined by one skilled in the art without undueexperimentation.

In a preferred embodiment of the invention, the adjuvant isimmunostimulatory oligonucleotides containing unmethylated CpGdinucleotides (“CpG”). CpGs are known in the art as being adjuvants whenadministered by both systemic and mucosal routes (WO 96/02555, EP468520, Davis et al., J. Immunol, 1998, 160(2): 870-876, McCluskie andDavis, J. Immunol., 1998, 161(9): 4463-6). CpG is an abbreviation forcytosineguanosinc dinucicotide motifs present in DNA. Historically, itwas observed that the DNA fraction of BCG could exert an anti-tumoureffect. In further studies, synthetic oligonucleotides derived from BCGgene sequences were shown to be capable of inducing immunostimulatoryeffects (both in vitro and in vivo). The authors of these studiesconcluded that certain palindromic sequences, including a central CGmotif, carried this activity. The central role of the CG motif inimmunostimulation was later elucidated in a publication by Krieg, 1995,Nature 374, p. 546. Detailed analysis has shown that the CG motif has tobe in a certain sequence context, and that such sequences are common inbacterial DNA but are rare in vertebrate DNA. The immunostimulatorysequence is often: Purine, Purine, C, G, pyrimidine, pyrimidine; whereinthe dinucleotide CG motif is not methylated, but other unmethylated CpGsequences are known to be immunostimulatory and may be used in thepresent invention.

CpG when formulated into vaccines, is generally administered in freesolution together with free antigen (WO 96/02555; McCluskie and Davis,supra) or covalently conjugated to an antigen (PCT Publication No. WO98/16247), or formulated with a carrier such as aluminium hydroxide((Hepatitis surface antigen) Davis et al. supra; Brazolot-Millan et al.,Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

Other supplementary immune potentiating agents, such as cytokines, maybe delivered in conjunction with the Fc-antigen fusion peptides of theinvention. The cytokines contemplated are those that will enhance thebeneficial effects that result from administering the immunomodulatorsaccording to the invention. Cytokines are factors that support thegrowth and maturation of cells, including lymphocytes. It is believedthat the addition of cytokines will augment cytokine activity stimulatedin vivo by carrying out the methods of the invention. The preferredcytokines are interleukin (IL)-1, IL-2, gamma-interferon and tumornecrosis factor α. Other useful cytokines are believed to be IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,erythropoietin, leukemia inhibitory factor, oncostatin-M, ciliaryneurotrophic factor, growth hormone, prolactin, CD40-ligand,CD27-ligand, CD30-ligand, alpha-interferon, beta-interferon, and tumornecrosis factor β. Other cytokines known to modulate T-cell activity ina manner likely to be useful according to the invention are colonystimulating factors and growth factors including granulocyte and/ormacrophage stimulating factors (GM-CSF, G-CSF and CSF-1) and plateletderived, epidermal, insulin-like, transforming and fibroblast growthfactors. The selection of the particular cytokines will depend upon theparticular modulation of the immune system that is desired. The activityof cytokines on particular cell types is known to those of ordinaryskill in the art.

The precise amounts of the foregoing cytokines used in the inventionwill depend upon a variety of factors, including the Fc-antigenselected, the dose and dose-timing selected, the mode of administrationand the characteristics of the subject. The precise amounts selected canbe determined without undue experimentation, particularly since athreshold amount will be any amount which will enhance the desiredimmune response. Thus, it is believed that nanogram to milligram amountsare useful, depending upon the mode of delivery, but that nanogram tomicrogram amounts are likely to be most useful because physiologicallevels of cytokines are correspondingly low.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a Fc-antigen fusion protein thatwill alone, or together with further doses, stimulate an immune responseas desired. This may involve the stimulation of a humoral antibodyresponse resulting in an increase in antibody titer in serum, improvedmucosal immunity, a clonal expansion of cytotoxic T lymphocytes ortolerance to an antigen, including a self antigen. It is believed thatdoses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram,depending upon the mode of administration, will be effective. Thepreferred range is believed to be between about 500 nanograms and 500micrograms/kilogram, and most preferably between 1 microgram and 100micrograms/kilogram. The absolute amount will depend upon a variety offactors, including the Fc-antigen fusion protein selected, the immunemodulation desired, whether the administration is in a single ormultiple doses, and individual patient parameters including age,physical condition, size and weight. For treatment of a subject with atumor the size, type, location and metastases of the tumor may befactored in when determining the amount of Fc-antigen to administer.These factors are well known to those of ordinary skill in the art andcan be addressed with no more than routine experimentation.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular Fc-antigen fusionprotein selected, the particular condition being treated and the dosagerequired for therapeutic efficacy. The methods of this invention,generally speaking, involve delivering the Fc-antigen fusion protein ofthe invention to an epithelial surface. Preferred modes ofadministration are oral, intrapulmonary, intrabinary and intranasal.

Compositions may be conveniently presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy. Allmethods include the step of bringing the Fc-antigen fusion protein intoassociation with a carrier which constitutes one or more accessory,ingredients. In general, the compositions are prepared by uniformly andintimately bringing the Fc-antigen fusion protein into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

The preferred amount of Fc-antigen fusion protein in all pharmaceuticalpreparations made in accordance with the present invention should be atherapeutically effective amount thereof which is also a medicallyacceptable amount thereof. Actual dosage levels of Fc-antigen fusionprotein in the pharmaceutical compositions of the present invention maybe varied so as to obtain an amount of Fc-antigen fusion protein whichis effective to achieve the desired therapeutic response for aparticular patient, pharmaceutical composition of Fc-antigen fusionprotein, and mode of administration, without being toxic to the patient.

The selected dosage level and frequency of administration will dependupon a variety of factors including the route of administration, thetime of administration, the rate of excretion of the therapeuticagent(s) including Fc-antigen fusion protein, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith Fc-antigen fusion protein, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated and thelike factors well known in the medical arts. For example, the dosageregimen is likely to vary with pregnant women, nursing mothers andchildren relative to healthy adults.

A physician having ordinary skill in the art can readily determine andprescribe the therapeutically effective amount of the pharmaceuticalcomposition required. For example, the physician could start doses ofFc-antigen fusion protein employed in the pharmaceutical composition ofthe present invention at levels lower than that required to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

The pharmaceutical compositions of the present invention, including theFc-antigen fusion protein to a therapeutic as the active agent aresuitable preferably for oral, sublingual, and intranasal delivery. Thepharmaceutical compositions are suitable for the delivery of theFc-antigen fusion protein to epithelial barriers. The pharmaceuticalcompositions may also be formulated to be suitable for parenteral,transdermal, intradermal and intravenous delivery.

The pharmaceutical compositions, containing biologically activeFc-antigen fusion protein as the active agent, that are suitable fortransmucosal delivery via oral cavity delivery are in the form of asolid as lingual, buccal or sublingual tablets, troches, (lozenges),powders, time-release granules, pellets or the like may also be used, orin the form of a liquid as a liquid drop or drops, aerosol spray ormist, applied sublingually (under the tongue), on top of the tongue, orbuccally (between the cheek and gingiva). The rate of oral mucosalmembrane absorption of Fc-antigen fusion protein, is controlled by thespecific liquid or solid dosage formulation selected. Specificformulations allow the process of absorption to take place over asustained, but relatively short period of time, allowing for a gradualbuild up and constant blood level of the Fc-antigen fusion protein.

For prolonged delivery, the active ingredient can be formulated as adepot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the Fc-antigen fusion protein.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle before useSuch liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound. By way of example, but not by limitation, theFc-antigen fusion protein may be conjugated to the followingtherapeutics for epithelial barrier targeted delivery:

For buccal or sublingual administration, the compositions may take theform of tablets or lozenges formulated in conventional manner. Forrectal and vaginal routes of administration, the active ingredient maybe formulated as solutions (for retention enemas) suppositories orointments.

For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The coordinate administration methods of the instant inventionoptionally incorporate effective mucolytic or mucus-clearing agents,which serve to degrade, thin or clear mucus from intranasal mucosalsurfaces to facilitate absorption of intranasally administeredbiotherapeutic agents. Within these methods, a mucolytic ormucus-clearing agent is coordinately administered as an adjunct compoundto enhance intranasal delivery of the biologically active agent.Alternatively, an effective amount of a mucolytic or mucus-clearingagent is incorporated as a processing agent within a multi-processingmethod of the invention, or as an additive within a combinatorialformulation of the invention, to provide an improved formulation thatenhances intranasal delivery of biotherapeutic compounds by reducing thebarrier effects of intranasal mucus.

Agents disclosed herein may be administered to subjects by a variety ofmucosal administration modes, including by oral, rectal, vaginal,intranasal, intrapulmonary, or transdermal delivery, or by topicaldelivery to the eyes, ears, skin or other mucosal surfaces. Optionally,fusion proteins, and other biologically active agents disclosed hereincan be coordinately or adjunctively administered by non-mucosal routes,including by intramuscular, subcutaneous, intravenous, intra-atrial,intra-articular, intraperitoneal, or parenteral routes. In otheralternative embodiments, the biologically active agent(s) can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a mammalian subject, for example as a component of anex vivo tissue or organ treatment formulation that contains thebiologically active agent in a suitable, liquid or solid carrier.

Compositions according to the present invention are often administeredin an aqueous solution as a nasal or pulmonary spray and may bedispensed in spray form by a variety of methods known to those skilledin the art. Preferred systems for dispensing liquids as a nasal sprayare disclosed in U.S. Pat. No. 4,511,069. Such formulations may beconveniently prepared by dissolving compositions according to thepresent invention in water to produce an aqueous solution, and renderingthe solution sterile. The formulations may be presented in multi-dosecontainers, for example in the sealed dispensing system disclosed inU.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systemshave been described in Transdermal Systemic Medication, Chien, Y. W.Ed., Elsevier Publishers, New York, 1985; and in U.S. Pat. No. 4,778,810(each incorporated herein by reference). Additional aerosol deliveryforms may include, e.g., compressed air-, jet-, ultrasonic-, andpiezoelectric nebulizers, which deliver the biologically active agentdissolved or suspended in a pharmaceutical solvent, e.g., water,ethanol, or a mixture thereof.

Nasal and pulmonary spray solutions of the present invention typicallycomprise the drug or drug to be delivered, optionally formulated with asurface active agent, such as a nonionic surfactant (e.g.,polysorbate-80), and one or more buffers. In some embodiments of thepresent invention, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution is optionally betweenabout pH 6.8 and 7.2, but when desired the pH is adjusted to optimizedelivery of a charged macromolecular species (e.g., a therapeuticprotein or peptide) in a substantially unionized state. Thepharmaceutical solvents employed can also be a slightly acidic aqueousbuffer (pH 4-6). Suitable buffers for use within these compositions areas described above or as otherwise known in the art. Other componentsmay be added to enhance or maintain chemical stability, includingpreservatives, surfactants, dispersants, or gases. Suitablepreservatives include, but are not limited to, phenol, methyl paraben,paraben, m-cresol, thiomersal, benzylalkonium chloride, and the like.Suitable surfactants include, but are not limited to, oleic acid,sorbitan trioleate, polysorbates, lecithin, phosphotidyl cholines, andvarious long chain diglycerides and phospholipids. Suitable dispersantsinclude, but are not limited to, ethylenediaminetetraacetic acid, andthe like. Suitable gases include, but are not limited to, nitrogen,helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbondioxide, air, and the like.

Within alternate embodiments, mucosal formulations are administered asdry powder formulations comprising the biologically active agent in adry, usually lyophilized, form of an appropriate particle size, orwithin an appropriate particle size range, for intranasal delivery.Minimum particle size appropriate for deposition within the nasal orpulmonary passages is often about 0.5u mass median equivalentaerodynamic diameter (MMEAD), commonly about 1u MMEAD, and moretypically about 2u MMEAD. Maximum particle size appropriate fordeposition within the nasal passages is often about 10u MMEAD, commonlyabout 8u MMEAD, and more typically about 4u MMEAD. Intranasallyrespirable powders within these size ranges can be produced by a varietyof conventional techniques, such as jet milling, spray drying, solventprecipitation, supercritical fluid condensation, and the like. These drypowders of appropriate MMEAD can be administered to a patient via aconventional dry powder inhaler (DPI) which rely on the patient'sbreath, upon pulmonary or nasal inhalation, to disperse the power intoan aerosolized amount. Alternatively, the dry powder may be administeredvia air assisted devices that use an external power source to dispersethe powder into an aerosolized amount, e.g., a piston pump.

Dry powder devices typically require a powder mass in the range fromabout 1 mg to 20 mg to produce a single aerosolized dose (“puff”). Ifthe required or desired dose of the biologically active agent is lowerthan this amount, the powdered active agent will typically be combinedwith a pharmaceutical dry bulking powder to provide the required totalpowder mass. Preferred dry bulking powders include sucrose, lactose,dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), andstarch. Other suitable dry bulking powders include cellobiose, dextrans,maltotriose, pectin, sodium citrate, sodium ascorbate, and the like.

To formulate compositions for mucosal delivery within the presentinvention, the biologically active agent can be combined with variouspharmaceutically acceptable additives, as well as a base or carrier fordispersion of the active agent(s). Desired additives include, but arenot limited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, etc. In addition, localanesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodiumchloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),solubility enhancing agents (e.g., cyclodextrins and derivativesthereof), stabilizers (e.g., serum albumin), and reducing agents (e.g.,glutathione) can be included. When the composition for mucosal deliveryis a liquid, the tonicity of the formulation, as measured with referenceto the tonicity of 0.9% (w/v) physiological saline solution taken asunity, is typically adjusted to a value at which no substantial,irreversible tissue damage will be induced in the nasal mucosa at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about ⅓ to 3, more typically ½ to 2, and mostoften ¾ to 1.7.

The biologically active agent may be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base may be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (e.g. maleic anhydride) with other monomers (e.g.methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymerssuch as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives such as hydroxymethylcellulose,hydroxypropylcellulose, etc., and natural polymers such as chitosan,collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metalsalts thereof. Often, a biodegradable polymer is selected as a base orcarrier, for example, polylactic acid, poly(lactic acid-glycolic acid)copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolicacid) copolymer and mixtures thereof. Alternatively or additionally,synthetic fatty acid esters such as polyglycerin fatty acid esters,sucrose fatty acid esters, etc. can be employed as carriers. Hydrophilicpolymers and other carriers can be used alone or in combination, andenhanced structural integrity can be imparted to the carrier by partialcrystallization, ionic bonding, crosslinking and the like. The carriercan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to the nasal mucosa. The use of a selected carrier in thiscontext may result in promotion of absorption of the biologically activeagent.

The biologically active agent can be combined with the base or carrieraccording to a variety of methods, and release of the active agent maybe by diffusion, disintegration of the carrier, or associatedformulation of water channels. In some circumstances, the active agentis dispersed in microcapsules (microspheres) or nanocapsules(nanospheres) prepared from a suitable polymer, e.g., isobutyl2-cyanoacrylate (see, e.g., Michael, et al., J. Pharmacy Pharmacol.43:1-5, 1991), and dispersed in a biocompatible dispersing mediumapplied to the nasal mucosa, which yields sustained delivery andbiological activity over a protracted time.

To further enhance mucosal delivery of pharmaceutical agents within theinvention, formulations comprising the active agent may also contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10000 and preferably not more than3000. Exemplary hydrophilic low molecular weight compound include polyolcompounds, such as oligo-, di- and monosaccarides such as sucrose,mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose,D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin andpolyethylene glycol. Other examples of hydrophilic low molecular weightcompounds useful as carriers within the invention includeN-methylpyrrolidone, and alcohols (e.g., oligovinyl alcohol, ethanol,ethylene glycol, propylene glycol, etc.). These hydrophilic lowmolecular weight compounds can be used alone or in combination with oneanother or with other active or inactive components of the intranasalformulation.

The compositions of the invention may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

Therapeutic compositions for administering the biologically active agentcan also be formulated as a solution, microemulsion, or other orderedstructure suitable for high concentration of active ingredients. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Proper fluidity for solutions can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of adesired particle size in the case of dispersible formulations, and bythe use of surfactants. In many cases, it will be desirable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe biologically active agent can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments of the invention, the biologically active agentis administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system or bioadhesive gel. Prolonged deliveryof the active agent, in various compositions of the invention can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.When controlled release formulations of the biologically active agent isdesired, controlled release binders suitable for use in accordance withthe invention include any biocompatible controlled-release materialwhich is inert to the active agent and which is capable of incorporatingthe biologically active agent. Numerous such materials are known in theart. Useful controlled-release binders are materials that aremetabolized slowly under physiological conditions following theirintranasal delivery (e.g., at the nasal mucosal surface, or in thepresence of bodily fluids following transmucosal delivery). Appropriatebinders include but are not limited to biocompatible polymers andcopolymers previously used in the art in sustained release formulations.Such biocompatible compounds are non-toxic and inert to surroundingtissues, and do not trigger significant adverse side effects such asnasal irritation, immune response, inflammation, or the like. They aremetabolized into metabolic products that are also biocompatible andeasily eliminated from the body.

Exemplary polymeric materials for use in this context include, but arenot limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolysable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids (PGA) and polylactic acids (PLA),poly(DL-lactic acid-co-glycolic acid) (DL PLGA), poly(D-lacticacid-coglycolic acid) (D PLGA) and poly(L-lactic acid-co-glycolic acid)(L PLGA). Other useful biodegradable or bioerodable polymers include butare not limited to such polymers as poly(ε-caprolactone),poly(ε-aprolactone-CO-lactic acid), poly(ε-aprolactone-CO-glycolicacid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate),hydrogels such as poly(hydroxyethyl methacrylate), polyamides,poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid andthe like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide),polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides,polysaccharides and copolymers thereof. Many methods for preparing suchformulations are generally known to those skilled in the art. J. R.Robinson, ed., Sustained and Controlled Release Drug Delivery Systems,Marcel Dekker, Inc., New York, 1978, incorporated herein by reference.Other useful formulations include controlled-release compositions suchas are known in the art for the administration of leuprolide (tradename: Lupron™), e.g., microcapsules, U.S. Pat. Nos. 4,652,441 and4,917,893, each incorporated herein by reference, lactic acid-glycolicacid copolymers useful in making microcapsules and other formulations,U.S. Pat. Nos. 4,677,191 and 4,728,721, each incorporated herein byreference, and sustained-release compositions for water-solublepeptides. U.S. Pat. No. 4,675,189, incorporated herein by reference.

The mucosal formulations of the invention typically must be sterile andstable under all conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the active compound in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The prevention of the action ofmicroorganisms can be accomplished by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

In more detailed aspects of the invention, the biologically active agentis stabilized to extend its effective half-life following delivery tothe subject, particularly for extending metabolic persistence in anactive state within the physiological environment (e.g., at the nasalmucosal surface, in the bloodstream, or within a connective tissuecompartment or fluid-filled body cavity). For this purpose, thebiologically active agent may be modified by chemical means, e.g.,chemical conjugation, N-terminal capping, PEGylation, or recombinantmeans, e.g., site-directed mutagenesis or construction of fusionproteins, or formulated with various stabilizing agents or carriers.Thus stabilized, the active agent administered as above retainsbiological activity for an extended period (e.g., 2-3, up to 5-10 foldgreater stability) under physiological conditions compared to itsnon-stabilized form.

In accordance with the various treatment methods of the invention, thebiologically active agent is delivered to a mammalian subject in amanner consistent with conventional methodologies associated withmanagement of the disorder for which treatment or prevention is sought.In accordance with the disclosure herein, a prophylactically ortherapeutically effective amount of the biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

Mucosal administration according to the invention allows effectiveself-administration of treatment by patients, provided that sufficientsafeguards are in place to control and monitor dosing and side effects.Mucosal administration also overcomes certain drawbacks of otheradministration forms, such as injections, that are painful and exposethe patient to possible infections and may present drug bioavailabilityproblems. For nasal and pulmonary delivery, systems for controlledaerosol dispensing of therapeutic liquids as a spray are well known. Inone embodiment, metered doses of active agent are delivered by means ofa specially constructed mechanical pump valve (U.S. Pat. No. 4,511,069,incorporated herein by reference). This hand-held delivery device isuniquely nonvented so that sterility of the solution in the aerosolcontainer is maintained indefinitely.

For prophylactic and treatment purposes, the biologically activeagent(s) disclosed herein may be administered to the subject in a singlebolus delivery, via continuous delivery (e.g., continuous transdermal,mucosal, or intravenous delivery) over an extended time period, or in arepeated administration protocol (e.g., by an hourly, daily or weekly,repeated administration protocol). In this context, a therapeuticallyeffective dosage of the biologically active agent(s) may includerepeated doses within a prolonged prophylaxis or treatment regimen, thatwill yield clinically significant results to alleviate one or moresymptoms or detectable conditions associated with a targeted disease orcondition as set forth above. Determination of effective dosages in thiscontext is typically based on animal model studies followed up by humanclinical trials and is guided by determining effective dosages andadministration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (e.g., immunologic and histopathologicassays). Using such models, only ordinary calculations and adjustmentsare typically required to determine an appropriate concentration anddose to administer a therapeutically effective amount of thebiologically active agent(s) (e.g., amounts that are intranasallyeffective, transdermally effective, intravenously effective, orintramuscularly effective to elicit a desired response). In alternativeembodiments, an “effective amount” or “effective dose” of thebiologically active agent(s) may simply inhibit or enhance one or moreselected biological activity(ies) correlated with a disease orcondition, as set forth above, for either therapeutic or diagnosticpurposes.

The actual dosage of biologically active agents will of course varyaccording to factors such as the disease indication and particularstatus of the subject (e.g., the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, etc.), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the biologicallyactive agent(s) for eliciting the desired activity or biologicalresponse in the subject. Dosage regimens may be adjusted to provide anoptimum prophylactic or therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental sideeffects of the biologically active agent is outweighed in clinical termsby therapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a biologically active agent withinthe methods and formulations of the invention is 0.01 ug/kg-10 mg/kg,more typically between about 0.05 and 5 mg/kg, and in certainembodiments between about 0.2 and 2 mg/kg. Alternatively, a non-limitingrange for a therapeutically effective amount of a biologically activeagent within the methods and formulations of the invention is betweenabout 0.001 pmol to about 100 pmol per kg body weight, between about0.01 pmol to about 10 pmol per kg body weight, between about 0.1 pmol toabout 5 pmol per kg body weight, or between about 0.5 pmol to about 1.0pmol per kg body weight. Dosages within this range can be achieved bysingle or multiple administrations, including, e.g., multipleadministrations per day, daily or weekly administrations. Peradministration, it is desirable to administer at least one microgram ofthe biologically active agent (e.g., one or more Fc-antigen fusionproteins), more typically between about 10 ug and 5.0 mg, and in certainembodiments between about 100 ug and 1.0 or 2.0 mg to an average humansubject. It is to be further noted that for each particular subject,specific dosage regimens should be evaluated and adjusted over timeaccording to the individual need and professional judgment of the personadministering or supervising the administration of the permeabilizingpeptide(s) and other biologically active agent(s).

Dosage of biologically active agents may be varied by the attendingclinician to maintain a desired concentration at the target site. Forexample, a selected local concentration of the biologically active agentin the bloodstream may be about 1-50 nanomoles per liter, sometimesbetween about 1.0 nanomole per liter and 10, 15 or 25 nanomoles perliter, depending on the subject's status and projected or measuredresponse. In an alternative example, a selected local concentration ofthe biologically active agent in the bloodstream may be between about0.1 pmol/L to about 1000 pmol/L of blood plasma or CSF, between about1.0 pmol/L to about 100 pmol/L of blood plasma or CSF, between about 1.0pmol/L to about 10 pmol/L of blood plasma or CSF, or between about 5.0pmol/L to about 10 pmol/L of blood plasma or CSF. Higher or lowerconcentrations may be selected based on the mode of delivery, e.g.,trans-epidermal, rectal, oral, or intranasal delivery versus intravenousor subcutaneous delivery. Dosage should also be adjusted based on therelease rate of the administered formulation, e.g., of a nasal sprayversus powder, sustained release oral versus injected particulate ortransdermal delivery formulations, etc. To achieve the same serumconcentration level, for example, slow-release particles with a releaserate of 5 nanomolar (under standard conditions) would be administered atabout twice the dosage of particles with a release rate of 10 nanomolar.

The instant invention also includes kits, packages and multicontainerunits containing the above described pharmaceutical compositions, activeingredients, and/or means for administering the same for use in theprevention and treatment of diseases and other conditions in mammaliansubjects. Briefly, these kits include a container or formulation thatcontains one or more Fc-antigen fusion proteins, in combination withmucosal delivery enhancing agents disclosed herein formulated in apharmaceutical preparation for mucosal delivery. The biologically activeagent(s) is/are optionally contained in a bulk dispensing container orunit or multi-unit dosage form. Optional dispensing means may beprovided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingthat the pharmaceutical agent packaged therewith can be used mucosally,e.g., intranasally, for treating or preventing a specific disease orcondition. In more detailed embodiments of the invention, kits includeone or more mucosal delivery-enhancing agents selected from: (a)aggregation inhibitory agents; (b) charge modifying agents; (c) pHcontrol agents; (d) degradative enzyme inhibitors; (e) mucolytic ormucus clearing agents; (f) ciliostatic agents; (g) membranepenetration-enhancing agents (e.g., (i) a surfactant, (ii) a bile salt,(iii) a phospholipid or fatty acid additive, mixed micelle, liposome, orcarrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound,(vii) a long-chain amphipathic molecule, (viii) a small hydrophobicpenetration enhancer; (ix) sodium or a salicylic acid derivative; (x) aglycerol ester of acetoacetic acid, (xi) a cyclodextrin orbeta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) achelating agent, (xiv) an amino acid or salt thereof, (xv) anN-acetylamino acid or salt thereof, (xvi) an enzyme degradative to aselected membrane component, (xvii) an inhibitor of fatty acidsynthesis, (xviii) an inhibitor of cholesterol synthesis; or (xix) anycombination of the membrane penetration enhancing agents of(i)-(xviii)); (h) secondary modulatory agents of epithelial junctionphysiology, such as nitric oxide (NO) stimulators, chitosan, andchitosan derivatives; (i) vasodilator agents; (j) selectivetransport-enhancing agents; and (k) stabilizing delivery vehicles,carriers, supports or complex-forming species with which thebiologically active agent is/are effectively combined, associated,contained, encapsulated or bound to stabilize the active agent forenhanced mucosal delivery.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Materials and Method for HSV-2 gD-Fc Vaccine.

Cells, antibodies, and virus. Inner Medullary Collecting Duct (IMCD)cell line and IMCD cells expressing rat FcRn were obtained from Dr. NeilSimister at Brandies University. Vero and Chinese hamster ovary (CHO-K)cells were purchased from American Tissue Culture Collection (ATCC).IMCD, Vero, and CHO cells were maintained in DMEM complete medium(Invitrogen Life Technologies) supplemented with 10 mM HEPES, 10% fetalbovine serum, 2 mM L-glutamine, nonessential amino acids, and penicillin(0.1 mg/ml)/streptomycin (0.292 mg/ml). Recombinant IMCD and CHO cellswere grown under 400 mg/ml of G418 if necessary. Cells from spleen orbone marrow were grown in complete RPMI 1640 medium. Herpes SimplexVirus-2 (HSV-2) strain 186 was from Dr. Lawrence Stanberry (ColumbiaUniversity, New York, N.Y.) and HSV-2 stocks were prepared by infectionof Vero cell monolayers at a multiplicity of infection (MOI) of 0.01.All cells and viruses were grown in a humidified atmosphere of 5% CO₂ at37° C. Affinity purified antibody for mouse FcRn was made as previouslydescribed (30). HRP-conjugated donkey anti-rabbit or rabbit anti-mouseantibody was purchased from Pierce (Rockland). Purified mouse IgG andchicken IgY was from Rockland Laboratories, and HRP-conjugated goatanti-mouse IgG1, IgG2a and IgG3 were from Southern Biotech. All DNAmodifying enzymes were purchased from New England Biolabs. PurifiedHSV-2 glycoprotein D was purchased from Meridian Life Science.

Expression of gD-Fc Fusion Proteins. The cDNA encoding the extracellulardomain of HSV-2 gD (SEQ ID NO:2) with the signal peptide, 1-25 aa,nucleotides 1-75 of SEQ ID NO:2, which will be cleaved off in the matureform of gD was amplified by PCR from a plasmid provided by Dr. PatriciaG. Spear (Northwestern University) using the primer pair(5′-cccaagcttaccatggggcgtttgacctccggcgtc-3′ (SEQ IDNO:3,5′-agatcccgagccacctcctcc ggacccacccccgcctgatccgcccgggttgctggggg-3′,SEQ ID NO:4). The antisense primer introduces an extension with fourteencodons for glycine and serine residues (GSGGGGSGGGGSGS, SEQ ID NO:1).The Fc-fragment of mouse IgG2a containing the hinge, an extended CH2 anda CH3 domain was amplified by RT-PCR from the OKT3 hybridoma usingprimer pair, forward primer: 5′-GGA TCA GGC GGG GGT GGG TCC GGA GGA GGTGGC TCG GGA TCT GAG CCC AGA GGGCCC A-3′ (SEQ ID NO:5) (Bolded letterrepresents the GS linker sequence), and reverse primer:5′-CCGGAATTCTCATTTACCCGGAGTC-3′SEQ ID NO:6). The mouse IgG2a Fc fragmentwas used because mouse IgG2a, but not IgG1, is capable of binding mouseFcgRI, a high affinity receptor for IgG. Similarly, the forward primerfor IgG2a Fc has complementary glycine and serine codons for gD. Amutant Fc (HQ310 and HN433), unable to bind mouse FcRn, was made byoligonucleotide site-directed mutagenesis (Clontech) and designated asan Fc/mut. To construct a nonlytic Fc fragment, oligonucleotidesite-directed mutagenesis was used to replace the C1q binding motifGlu318, Lys320, Lys322 with Ala residues (14) (SEQ ID NO:7). Fusionswere then performed in a PCR-based gene assembly approach by mixing thecDNA for gD and the Fc fragment. All these DNA fragments were ligatedinto the pcDNA3 vector. Each construct was verified by DNA sequencing.

The plasmids containing the chimeric gD-Fc fragment (SEQ ID NO:8) weretransfected into CHO cells. G418-resistant clones were selected forsecretion of gD-Fc fusion protein. SDS-PAGE and Western blot wereperformed to assess the recombinant fusion proteins in serum-free medium(Invitrogen). The highest secreting clones were screened. Recombinantproteins were purified from CHO cell supernatants by affinitychromatography using Protein A Sepharose 4 Fast Flow (Amersham) or goatanti-mouse IgG affinity column (Rockland). Protein concentration wasmeasured with Coomassie (Bradford) protein assay kit (Pierce) usingmouse IgG2a as standard.

Western blot and SDS-PAGE gel electrophoresis. The proteins or celllysates were resolved on a 12% SDS-PAGE gel under a reducing ornon-reducing condition. Proteins were transferred onto a nitrocellulosemembrane (Schleicher & Schuell). The membranes were blocked with 5%non-fat milk, probed separately with anti-gD, anti-IgG Fc Ab oranti-mouse FcRn for 1 hr, and followed by incubation with HRP-conjugatedrabbit anti-mouse or donkey anti-rabbit Ab. All blocking, incubation,and washing were performed in PBST solution (PBS and 0.05% Tween 20).Proteins were visualized by ECL (Pierce).In vitro and in vivo transcytosis. The in vitro IgG transport assay wasperformed as a modification from previously-described methods (15, 36).IMCD cells expressing rat FcRn were grown on transwell filter inserts(Corning Costar) to form a monolayer exhibiting transepithelialelectrical resistances (TER, 300 Ω·cm²). TER was measured using atissue-resistance measurement equipped with planar electrodes (WorldPrecision Instruments). Monolayers were equilibrated in Hanks' balancedsalt solution. Fusion proteins (50 ug/ml) were applied to the apicalcompartment, and incubated with DMEM medium supplied with or without 1mg/ml of mouse IgG or chicken IgY as competitors for 2 hr at 37° C.degree. Transported proteins were sampled from the basolateral chamberand analyzed by reducing SDS-PAGE and Western blot-ECL. For in vivotransport, the biotinylated 20 ug of fusion proteins or gD alone in 20ul of PBS were administered intranasally (i.n.) into the mice that wereanethesitized with 100 ul of avertin (40 mg/ml). 8 hr later or atindicated time points, transported proteins in sera were determined byELISA.Mouse immunization and virus challenge. Six to eight week-old Femaleinbred C57BL/6 mice were purchased from Charles River. FcRn knockoutmice on a C57BL/6 background (16) were from the Jackson Laboratory. Allmice were housed in the animal resources facility at the University ofMaryland. All animal studies were reviewed and approved by theInstitutional Animal Care and Use Committee. To overcome the possiblemucosal immune tolerance (37, all proteins and PBS were mixed withimmunostimulatory DNA rich in CG motifs CpG ODN 1826 (abbreviated CpG).Groups of 5 mice were intranasally immunized with 20 μl of 20 uggD-Fc/wt, gD-Fc/mut, or recombinant gD alone in combination with 20 mgCpG (5′-TCCATGACGTTCCTGACGTT-3′, SEQ ID NO:9) (Invivogen) perimmunization at weeks 0 and 2 with an intraperitoneal injection of 100ul of avertin (40 mg/ml). An additional group of 5 mice wasmock-immunized with PBS following the same schedule. Mice were kept ontheir backs under anesthesia to allow the inoculum to be taken up.

Mice were inoculated with viruses intravaginally as described previously(27, 38). Briefly, prior to inoculation, each mouse was subcutaneouslytreated with 3 mg of medroxyprogesterone acetate (Depo-Provera) 10 daysprior to virus inoculation. Hormonal pretreatment was necessary toinduce susceptibility of mice to genital HSV-2 inoculation, which mayreflect thinning of the genital epithelium or induction of the HSV entryreceptor, nectin-1, on vaginal epithelial cells. Mice anesthetized byavertin (40 mg/ml, Sigma) were infected intravaginally with 1×10⁴pfu/100 ml of HSV-2 strain 186. Mice were kept on their backs under theinfluence of anesthesia for 45 min to allow infection. Mice weremonitored for 15 days for the disease and death. Mice exhibiting severedisease symptom were euthanized. For virus titration, virus wasinoculated into Vero cells and incubated for 45 min at 37° C. The cellswere washed and DMEM containing 0.8% methcellulose and 2% FBS was addedto overlay the cells. The cells were cultured for 3 days, the overlaywas removed, and the cells were fixed with 3.7% formaldehyde for 1 hr,and stained with 1% crystal violet.

Preparation of single-cell suspensions from lymph nodes, spleen, lung,and vaginal tissues. Spleens and lymph nodes (39) were made intosingle-cell suspensions by passage through a sterile mesh screen. Cellswere resuspended in Hanks' balanced salt solution (HBSS) and counted bytrypan blue dye exclusion. For each experiment, cells were generallypooled from 3-5 mice in each group. For preparation of single-cellsuspension from the lung, mice were anesthetized with 400 ul of avertin(40 mg/ml) by i.p. injection. Lungs were perfused with 10 ml PBS throughthe right ventricle, removed, minced with blades, and incubated withHBSS containing 2.5 mM HEPES and 1.3 mM EDTA at 37° C. for 30 min,followed by treatment at 37° C. for 1 hr with 2.5 mg/ml collengase D(Roche) in RPMI 1640 medium containing 5% FBS. The resulting cells werefiltered through a 70-mm cell strainer (BD) and used for FACS analysis.

For isolation of vaginal cells, the vagina was excised, cutlongitudinally, and minced with a sterile scalpel in complete RPMI 1640culture medium. Minced tissues (epithelium and lamina propria) weredigested in complete medium with sterile 0.25% collagenase D (Sigma).Digestion was accomplished with shaking incubation at 37° C. for 30 min.After digestion, tissues and cells were filtered through a sterile gauzemesh and washed with RPMI 1640 medium, and additional tissue debris wasexcluded by slow-speed centrifugation for 1 min. Cells were collectedfrom the supernatant by centrifugation, resuspended in HBSS, and countedby trypan blue dye exclusion.

Flow cytometry. Single cell suspensions from the spleen, lung or vaginaltissues were collected and cells were spun down. Erythrocytes were thenlysed in 0.14 M NH₄C1, 0.017 M Tris-HCl at pH 7.2 on ice for 5 min.Cells were preincubated with an Fc block (mAb to CD16-CD32, 2.4G2,PharMingen) and washed in FACS buffer (HBSS, 2% bovine serum albumin,0.01% sodium azide). Cells were incubated with specific antibody (0.25ug/10⁶ cells/100 ul) directly conjugated to fluorsecein isothiocyante(FITC), phycoerythrin (PE), washed, transferred to FACS buffer, andanalyzed using a FACSAire (Becton Dickinson, Mountain View, Calif.) andFlowJo software (Tree Star). The mAbs (PharMingen) we used wereanti-CDR, 500A2; anti-CD4, RM4-5; anti-CD8, 53-6.7; anti-IFN-γ, XMG1.2;anti-B220, RA3-6B2; FAS, Jo2; CD19, 1D3. PNA was from Sigma. PurifiedHSV-2 gD proteins were labeled with Alexa Fluoro647 protein labeling kit(Invitrogen) according to the manufacture's instruction. Cells incubatedwith isotype control antibodies were used to determine the backgroundfluorescence. The isotype control antibodies included in each experimentwere considered the true baseline fluorescence used to evaluate andillustrate the results for the cell-specific antigen markers.T cell proliferation. Single cell suspensions from mouse spleen weresuspended in RPMI-1640 with 1% FCS, 2.5 mM Hepes at 10⁷/ml.Carboxyfluorescein diacetate succinimidyl ester (CFSE, 5 mM stock,Invitrogen) was 10-fold diluted with PBS, 4 ul of diluted CFSE was thenadded into 10⁷/ml cells for a 2 uM final concentration. The reaction wasincubated for 10 min at 37° C. Cold FBS (1 ml) was added and incubatedon ice for 5 min to stop the reaction. The cells were washed twice withRPMI-1640 with 10% FCS. Labeled cells (5×10⁵) were plated into 96 wellplates in 200 ul of medium and cultured for 5 days. The cells were thenharvested and subjected to flow cytometry assay.Intracellular cytokine staining. Intracellular IFN-γ production byprimed CD4⁺ and CD8⁺ T cells was evaluated using bulk splenocytes orisolated lung or vaginal infiltrating lymphocytes incubated for 4 hrwith 25 ug/ml of the purified gD protein or medium alone. Cells werethen cultured for another 6 hr in the presence of brefeldin A (Sigma).The cells were then washed and incubated with anti-CD16/CD32 antibody toblock Fcγ receptors, and stained with anti-mouse CD4, CD8, and CD3antibodies for 15 min at 4° C. After fixation and membrane penetrationwith Cytofix/Cytoperm Plus (BD Biosciences), cells were stained forintracellular IFN-γ for 30 min on ice. Cells were washed three times,resuspended in FACS buffer, and analyzed by flow cytometry.Enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbentspot (ELISPOT), and neutralization test. For the detection ofgD-specific antibodies in serum, bronchial lavage and vaginal fluid,high-binding ELISA plates (Maxisorp, Nunc) were coated with 5 μg/ml ofrecombinant gD protein in PBS and incubated overnight at 4° C. Plateswere then washed three times with 0.02% Tween 20 in PBS and blocked with1% BSA in PBS for 1 hr at room temperature. Samples were seriallydiluted in 0.25% BSA-PBS and incubated for 2 hr at room temperature.HRP-conjugated rabbit anti-mouse IgG antibody (1:2,000, Pierce) oranti-mouse subclass-specific antibodies (1:5000, SouthernBiotech) wasadded and followed by colorimetric assay using substrate tetramethylbenzidine and a Victor III microplate reader (Perkin Elmer). Titersrepresent the highest dilution of samples showing a 2-fold OD₄₅₀ valueover controls. Neutralizing antibodies were measured by a standard virusneutralization assay. Sera were heat-inactivated, diluted 10-fold, thenin two-fold steps in MEM with 2% FBS. Fifty PFU of HSV-2 was added perwell and incubated at 37° C. for 1 hr. Then, plaque assays wereperformed. The titers were expressed as the reciprocal of the twofoldserial dilution preventing the appearance of the CPE. Each assay wasdone in triplicate.

For measuring gD-specific antibody-producing plasma cells, the 96-wellELISPOT plates (Millipore) were coated with 5 ug/ml gD and blocked withRPMI 5% FCS (Invitrogen) for 90 min at 37° C. and 5% CO₂. Serialdilutions of bone marrow single-cell suspensions were prepared in RPMIand incubated in the coated wells for 24 hr at 37° C. in 5% CO₂. Cellswere removed, and plates were washed 5 times with 0.1% Tween 20 in PBS,then incubated with biotin labeled goat anti-mouse IgG-specific antibody(1:1500, Sigma) for 2 hr. After washing the cells, with PBS, the avidinconjugated HRP (1:2,000, Vector Laboratories) was added and incubatedfor 1 hr, followed by substrate from the AEC kit (BD Biosciences). Spotswere counted with ELISPOT Reader and analyzed with software (Zesis).Mouse cytokines IFN-γ, IL-2, and IL-4 from the cell culture supernatantwere analyzed by ELISA according to the manufacturer's instructions (BDBiosciences).

Immunofluorescence. Immunofluorescence was performed as previouslydescribed (40). Briefly, frozen serial sections of tissues werecold-fixed in acetone for 3 min at 4° C. and blocked with 10% normalgoat serum and stained with biotin-PNA (germinal centers, red), Alexa647-IgD or anti-B220 (B cells, green), followed by Alexa 555-avidin or488 Fluoro-conjugated IgG of the corresponding species. After each step,cells were washed at least three times with 0.1% Tween-20 in PBS. Coverslips were mounted on slides with Prolong™ intifada kit (MolecularProbes) and examined using a Zees LSM 510 confocal fluorescencemicroscopy. Images were processed by Adobe Photoshop 7.0.Passive transfer of immune sera. Sera were collected from 3-5 mice pergroup 4 weeks after the immunization, then pooled, heat inactivated andstored frozen at −80° C. until use. Mice received a singleintraperitoneal (i.p.) injection of 0.3 ml immune sera 3 days prior tochallenge to allow distribution and equilibration of antibody to alltissues prior to virus inoculation. Mice were challenged intravaginallywith 1×10⁴ PFU HSV-2 186 strain.Statistics analysis. To compare survival curves, Kaplan-Meier log-rankanalyses were used. Antibody titers, serum gD concentration, cytokineconcentration and virus titers were assessed by using the unpairedtwo-tailed t test. Graph Pad Prism 5 provided the software for thestatistical analysis.

Example 1

FcRn-mediated mucosal vaccine delivery, if feasible, may allow the hostto specifically sample an Fc-fused subunit vaccine in the mucosal lumen,followed by transport of an intact antigen across the mucosal epithelialbarrier. To test this possibility, we used a model pathogen herpessimplex virus type-2 (HSV-2), which causes sexually-transmitted diseaseand initiates infection primarily at the mucosa of the genital tract(4). The development of HSV-2 subunit vaccine is mainly focused on itsmajor envelope glycoprotein D (gD), because of its key role in the earlysteps of viral infection and its being major target for both humoral andcellular immunity. Therefore, in this study, we determined the abilityof FcRn to deliver the model antigen, HSV-2 gD that plays a key role inthe early steps of viral infection and its being major target for bothhumoral and cellular immunity, across the mucosal barrier and furtherdefine protective immune responses and mechanisms relevant to this modeof mucosal vaccine delivery.

Example 2

FcRn can efficiently transport intact subunit vaccine antigens acrossthe respiratory mucosal barrier. To target gD to FcRn, we firstgenerated the fusion protein, gD-Fc/wt, by cloning the extracellulardomain of HSV-2 gD in frame with a modified form of the mouse IgG2a Fcfragment (FIG. 1). We also generated a similarly modified gD-Fc/mutfusion protein that cannot not bind FcRn owing to H to A substitutionsat positions 310 and 433 (13). In both cases, the complement C1q-bindingmotif was eliminated to abrogate C1q binding (14) (data not shown).Comparison of these fusion proteins allowed us to evaluate theefficiency of FcRn-mediated transport and immunization efficacy. Toascertain whether the gD-Fc/wt but not the gD-Fc/mut fusion proteinswere transported by FcRn, two criteria were applied. First, IMCD cellsexpressing FcRn (15) were evaluated for their ability to transport gD-Fcproteins in a transwell model. Indeed, FcRn-dependent transcytosis ofintact gD-Fc/wt was detected in IMCD cells expressing FcRn (15) (datanot shown). Second, we determined whether the gD-Fc/wt reached thebloodstream after intranasal (i.n.) inoculation. FcRn expression inmouse trachea and lung and its absence in the adult intestine wereconfirmed. To investigate the ability of gD-Fc/wt, gD-Fc/mut and gDproteins to undergo mucosal transport in vivo, 20 ug of the theseproteins were administered i.n. and their presence in serum was measured8 hr later using an ELISA. In comparison with gD and gD-Fc/mut, gD-Fc/wtprotein was abundantly detected in the sera of FcRb WT mice. Dependenceon FcRn was further confirmed by the finding that the serumconcentrations of gD-Fc/wt after administration into FcRn knockout (KO)mice (16) was greatly reduced as compared with that observed in FcRn WTmice (Data not shown). These results showed that efficient delivery ofgD across the respiratory barrier to the bloodstream was dependent onthe Fc moiety and its ability to interact with FcRn.

Example 3

Engagement of FcRn greatly increased the efficiency by which gD antigensinduced antigen-specific antibody and cellular immune responses. We thendetermined whether FcRn-dependent transport augmented immune responsepotential of the gD protein. Mice were immunized i.n. with the gD-Fc, gDprotein, or PBS, all in combination with CpG, and boosted after 2 wks.The gD unlinked to an Fc fragment allowed us to evaluateFcRn-independent effects in vivo, and also determine the magnitude ofany observed enhancement in immune responses conferred by inclusion ofthe Fc and FcRn engagement. Given the tolerogenic potential of at leastsome immature dendritic cells (DCs) in vivo, we co-administrated CpG, aligand for Toll-like receptor 9 (TLR-9), in an effort to overcomepossible mucosal tolerance (17). In a different study, we found thatco-administration of CpG ODN with an Fc fusion protein was required inorder to induce a robust IgG response (unpublished data). We firstmeasured antibody responses to gD in vaccinated animals at various timepoints up to 56 days. Significantly higher titers of IgG were seen inthe gD-Fc/wt immunized mice when compared with the gD, gD-Fc/mut,gD-wt/KO immunized and PBS-treated groups (FIG. 2A). Moreover, sera fromthe gD-Fc/wt immunized mice exhibited strong neutralizing activity (FIG.2B) and were able to most efficiently protect from intravaginal (ivag)challenge after passive transfer as compared with sera from all othergroups (Data not shown). Likewise, gD-Fc/wt proteins induced strongIFN-γ-producing CD8⁺ and CD4⁺ T cell responses, as evidenced bysignificantly higher frequency of IFN-γ producing CD4⁺ (FIG. 2C) andCD8⁺ (FIG. 2C) T cells in response to gD stimulation in the spleens ofFcRn WT mice immunized with gD-Fc/wt relative to the other groups. Thecytokine responses were dominated by IFN-γ and IL-2 and with a lack ofIL-4 production (FIG. 2D). This dominant Th1 cytokine response was alsosupported by a major IgG2a subclass in the sera of the immunized mice(data not shown). It remains uncertain whether this polarized T cellresponse is caused by mucosal immunization as the result of FcRntargeting or, more likely, by the CpG used in as adjuvant, as CpGfavorably induces Th1-type immune responses (17).

It is important to note that an important consequence of FcRn-targetedmucosal delivery of subunit vaccine was to elicit a strong mucosalimmune response. This result is strongly supported by several lines ofevidence. First, the nasopharynx-associated lymphoid tissue (NALT) andthe mediastinal lymph nodes (MeLNs) draining the lung are usually thesite where mucosal immune responses are initiated against antigensadministered intranasally and reaching the lung (18). To ascertain theability of the FcRn-targeted mucosal immunization to induce localhumoral immune responses, we monitored the germinal center (GC) reactionin the MeLNs and spleens 10 days after the boost. Activated GCs arecharacterized by the presence of peanut agglutinin (PNA)-positive areasand high-level expression of Fas apoptotic death receptor in activated Bcells (19). As shown in FIG. 3A, the gD-Fc/wt immunization induced asubstantially higher of FAS⁺PNA⁺B220⁺ B cells in the MeLN or spleen incomparison with the percentages of other groups which was furthervalidated by immunofluorescence imaging (FIG. 3B). In addition, the GCstructure in the MeLNs in WT mice immunized by the gD-Fc/wt proteins wassustained much longer in comparison with that of other groups of theimmunized animals (FIG. 3C). Administration of CpG alone failed toelicit appreciable GCs in the draining MeLNs, spleens, or lungs (datanot shown). The formation and maintenance of GC generally leads to thedifferentiation of memory B cells and long-lived plasma cells. We alsoexamined the mesenteric, cervical and inguinal lymph nodes and we failedto detect GC formation or the significant number of IFNγ secreting Tcells at those sites. Second, it has been shown that i.n. administratedantigens can induce bronchus-associated lymphoid tissue (iBALT) in thelung, which is similar to GC structures (19). As shown in FIG. 3D, suchGC-like structures could be detected in the lungs of gD-Fc/wt, but notgD-Fc/mut immunized mice or other groups of immunized animals byco-localization of PNA- and B220-positive signals. It was also absentfrom other groups of immunized animals. Appearance of the GC in theiBALT in the lung is best explained by local induction due to thepresence of the transported gD antigen in the MeLNs and lung, againemphasizing that the MeLNs play a major role as the inductive site.Third, antibodies, in particular IgA and IgG, represent the firstdefense line on mucosal surfaces. In order to assess the ability ofFcRn-targeted mucosal immunization to induce gD-specific antibody inmucosal secretions, bronchial alveolar lavage (BAL) and vaginal washeswere collected 10 days following the boost and analyzed for gD-specificIgG and IgA by ELISA. Significantly increased levels of gD-specific IgGwere present in the BAL and vaginal washes (data not shown) of thegD-Fc/wt protein immunized mice. WT, but not FcRn KO, mice that receivedthe gD-Fc/wt had high levels of gD-specific IgG in BAL and vaginalwashes (p<0.01, data not shown), suggesting the induction of mucosal IgGis FcRn-mediated. Of note is the observation that BAL- or vaginal IgGlevels were much higher than that of IgA, despite high IgA levels in thenasal washings (data not shown). Indeed, IgG appears a major isotype ofimmunoglobulin in the lower respiratory tract and reproductive tract.Fourth, with respect to T cell immune responses in the lung and MeLNs, 4days after the boost we detected significantly higher frequency of IFN-γproducing CD4⁺ and CD8⁺ T cells (FIGS. 3E and 3F) in response to gDstimulation in the mice immunized with the gD-Fc/wt in comparison withother groups. We conclude that antigen targeting to FcRn combined withthe mucosal adjuvant CpG produced strong antibody as well as T cellmucosal immune responses. Considering that an efficient protectivevaccine should induce immunity in the mucosa in order to hinder pathogenpenetration and spreading, the data presented here suggest thatFcRn-targeted mucosal immunization may provide an efficient approach forthe development of protective mucosal vaccines.

Preferably, an effective mucosal subunit vaccine should also elicit bothhumoral and cell-mediated immunity not only at the mucosal deliverysite, but also in systemic compartments that can access mucosal tissuesdistant from the immunization site (1-3). Studies have found thatmucosal immunization using the intranasal route is effective forgenerating antibody and T cell immune responses in the female genitaltract (1,2,4). Perhaps intranasal immunization stimulates cells in theNALT and its draining lymph nodes, leading to the migration of antibodysecreting cells and T cells generated in the airway into the genitaltract (20). We ivag challenged immunized mice with a lethal dose ofHSV-2 186 strain 4 weeks following the boost. As expected, all PBStreated control mice succumbed to lethal infection. All FcRn wt miceimmunized with the gD-Fc/wt survived with no obvious symptoms (data notshown), while gD-Fc/wt-immunized FcRn KO mice were incompletelyprotected (FIG. 4A). These data indicate that full protection isdependent on FcRn. Additionally, virus titers measured in the vaginalwashes showed that the gD-Fc/wt immunized mice had eliminated the virusby day 4 after challenge (FIG. 4B). In contrast, the other groups ofmice essentially failed to control viral replication. Several immunemechanisms may account for the protection of the distal vaginal mucosa.First, both mucosal and systemic antibody responses may play animportant protective role. This conclusion was supported by evidencethat the sera passively transferred from the gD-Fc immunized miceconferred a high level of protection (data not shown) and a significantamount of gD-specific IgG appeared in the vaginal secretions (data notshown). IgG is a major protective antibody in mouse vaginal secretionsafter immunization with attenuated HSV-2 (21). Second, T lymphocyteswere present in the vaginal epithelium of HSV-2 challenged mice at atime coincident with virus clearance. IFN-γ is clearly indispensable forresistance to HSV-2 infections (20). The gD-Fc/wt protein induced asignificantly higher frequency of IFN-γ producing CD4+ and CD8+ T cellsin the vaginal tissues from the challenged mice (FIG. 4C) in comparisonwith other groups. The strong T cell response induced by FcRn targetedimmunization could also provide resistance through direct lysis of MHCclass I or II-bearing infected epithelial cells. Overall these resultsdemonstrate that FcRn targeted mucosal immunization efficiently inducedprotective immunity.

Example 4

FcRn targeted mucosal delivery of vaccine engendered an effective memoryimmune response. Immunological memory is characterized by increasedlevels of effector T and B cells and by the host's ability to respondfaster and more vigorously to a second encounter with the pathogen orvaccine antigen (22, 23). Hence, an important criterion for any vaccineis the formation and maintenance of a reservoir of memory lymphocyteswith both adequate size and quality to maintain efficient immunesurveillance for prolonged periods. Immunological memory (23) has been aconcern in protein-based subunit mucosal vaccine development becausepreparations elicited levels of immunity immediately after vaccinationbut that immunity waned rapidly over time. However, a striking featurein this study is that FcRn targeted mucosal immunization promoted andsustained high levels of gD-specific plasma cells and memory B and Tcells at least 6 months after the boost. This conclusion is stronglysupported by several lines of evidence. First, gD-Fc/wt immunized micedeveloped significantly higher numbers of splenic memory B cellsresponsive to gD stimulation 6 months after the boost (FIG. 5A). ByELISPOT, we also found the significantly higher number of gD-specificIgG secreting plasma cells in the bone marrow of mice immunized withgD-Fc/wt in comparison with that of other groups (FIG. 5B). It remainsto be determined if gD-specific IgA-secreting plasma cells also develop.Second, higher titers of gD-specific IgG were maintained for six months,the latest point tested after a boost in the mice immunized with thegD-Fc/wt, but high titers were not observed in groups that did notenable FcRn trafficking (FIG. 5C). Both the increase in GC and memory Bcells in the spleen and the existence of long-lived plasma cells in thebone marrow niche can account for the maintenance of high levels ofgD-specific IgG in circulation. Third, an important feature of memory Tcells as opposed to the effector population is their proliferativepotential upon reencounter with an antigen. We detected significantnumbers of CD4⁺ (FIG. 5D, upper panel) and CD8⁺ (FIG. 5D, bottom panel)memory T cell proliferation by CFSE in response to gD recall in the miceimmunized with gD-Fc/wt, but not in any of the other groups. This resultwas also verified by significantly increased numbers of activelyproliferating T cells over time (data not shown). These data indicatethat the gD-specific T cells had maintained a significant proliferativepotential at least 6 months after the boost. Although the reason for thehigh memory T cell activity is not completely clear, IL-2-producing Tcells formed in the responding T cell population (data not shown) may beimportant since IL-2 plays an important role in the successful long-termsurvival of memory T cells in vivo (24). Fourth, to test if the memoryimmune response elicited from FcRn targeted mucosal immunization couldprovide protection, we again ivag challenged the immunized mice with alethal dose of HSV-2 186 strain 6 months after the boost. Mice immunizedwith the gD-Fc/wt proteins exhibited less severe disease symptoms (datanot shown) and had 80% survival (FIG. 5E), while the majority of mice inother groups succumbed to lethal mucosal infection.

Overall, this study shows that the FcRn/IgG transport pathway can beexploited to greatly enhance the efficacy of mucosally administeredvaccines. Previous studies have taken advantage of Fc fusion proteins toaugment the T cell immune response to myelin basic protein (25) andmucosally administered inactivated Francisella tularensis/antibodyimmune complexes have been shown enhance protection against the highlyvirulent strain of F. tularensis (26). We have shown that FcRn targetedmucosal immunization differs notably between WT and FcRn KO mice or thegD-Fc/wt and the gD-Fc/mut immunized mice in terms of mucosal andsystemic immune responses, cytokine expression profiles, the maintenanceof T and B cell memory and long lived bone marrow plasma cells, andresistance to infection. We established this principle using a modifiedform of the mouse IgG2a Fc fragment to facilitate the vaccine antigendelivery across mucosal the barrier. We are determining the efficiencyof FcRn dependent delivery of mucosal vaccines using other subclasses ofIgG, for example IgG1, IgG2b, and IgG3.

Earlier studies have shown intranasal and intravaginal immunization withgD or gB proteins in combination with CpG elicited immune responses andconferred partial protection to subsequent vaginal challenge with HSV-2(27-29). Lindqvist et al. (30) showed better protection, however, thesemice were intranasally or intravaginally immunized three times by gDplus α-galactosylceramide. The mechanism by which “plain” gD or gBproteins crossed the airway or female genital epithelial barrier inthose studies is not clear. Conditions that might explain passivemucosal transfer include the treatment of mice with agents that couldcompromise the integrity of the alveolo-capillary or mucosal epithelialbarrier (31, 32), such use of volatile anesthetics or 0.5% Tween inα-galactosylceramide preparation (27-30). Moreover, injections withDepo-Provera before vaginal immunization (28-30) may affect tightness ofthe vaginal epithelial cells because Provera is a long-actingprogesterone formulation and induces a diestrus-like state in genitaltract of female mice (4). In agreement with a previous report (33), ourother studies on examinations of the effect of CpG on protein transportacross the nasal/tracheal mucosa (unpublished data) are not consistentwith an effect of CpG on mucosal permeability or passive transfer.Regardless of the mechanism by which plain vaccine antigens may crossthe mucosal barrier, our results clearly document the benefits of FcRntargeting to maximize the potential of mucosally administered vaccinesto counteract mucosal pathogens, without the need for agents that maydamage or otherwise compromise the integrity of mucosal barriers.

We suggest, without being bound to any theory, the following model forFcRn-targeted immunization (FIG. 5F). In general, mucosal DCs captureantigens in mucosal-associated lymphoid tissues, and subsequentlymigrate to draining lymph nodes where they can prime T cells (33, 34)and initiate the cognate B cell response. Persistence of vaccineantigens can facilitate long-term memory immune responses (22, 23).Thus, while FcRn-mediated transport is necessary for efficient vaccineproven necessary for effective mucosal immunization, the ability of FcRnto protect gD-Fc/wt proteins from degradation may further support thedevelopment of systemic immunity by increasing the persistence ofgD-Fc/wt in circulation. Furthermore, this same protection property mayaugment long-term humoral immunity by maintaining serum high levels ofIgG antibodies specific for gD-Fc/wt. It remains to be determinedwhether this delivery method can augment pre-existing immunity. Takentogether, these results suggest that FcRn-targeted mucosal immunizationcould prove to be an effective strategy for maximizing the efficacy ofvaccinations directed against a broad range of mucosal pathogens.

The following references were cited in Examples 1-4.

-   1. Neutra, M. R. & Kozlowski, P. A, Nat. Rev. Immunol. 6, 148-158    (2006).-   2. Holmgren, J. & Czerkinsky, C., Nat. Med. 11(4 Supply, S45-53    (2005).-   3. McGhee, J. R. et al., Vaccine 10, 75-88 (1992).-   4. Gallichan, W. S. & Rosenthal, K. L., J. Infect. Dis. 177,    1155-1161 (1988).-   5. Neutra, M. R., et al., Nat. Immunol. 2, 1004-1009 (2001).-   6. Nochi, T. et al., J. Exp. Med. 204, 2789-2796 (2007).-   7. Ghetie, V. & Ward, E. S., Annu. Rev. Immunol. 18, 739-766 (2000).-   8. He, W. et al., Nature 455, 542-546 (2008).-   9. Dickinson, B. L. et al., J. Clin. Invest. 104, 903-911 (1999).-   10. Roopenian, D. C., & Akilesh, S., Nat. Rev. Immunol. 7, 715-725    (2007).-   11. Baker, K. et al., Semin. Immunopathol. 223-236 (2009).-   12. Yoshida, M. et al., J. Clin. Invest. 116, 2142-2151 (2006).-   13. Kim, J. K. et al., Eur. J. Immunol. 24, 2429-2434 (1994).-   14. Duncan, A. R. & Winter, G., Nature 332, 738-740 (1988).-   15. McCarthy, K. M., et al., J. Cell Sci. 113, 1277-1285 (2000).-   16. Roopenian, D. C. et al., 170, 3528-3533 (2003).-   17. van Duin, D. et al., Trends Immunol. 27, 49-55 (2006).-   18. Wolf, A. J. et al., J. Exp. Med. 205, 105-115 (2008).-   19. Moyron-Quiroz, J. E. et al., Nat. Med. 10, 927-934 (2004).-   20. Milligan, G. N. et al., Virology 318, 507-515 (2004).-   21. Parr, E. L. & Parr, M. B., J. Virol. 71, 8109-9115 (1997).-   22. Ahmed, R. & Gray, D., Science 272, 54-60 (1996).-   23. Bernasconi, N. L. et al., Science 298, 2199-2202 (2002).-   24. Dooms, H. et al., J. Exp. Med. 204, 547-557 (2007).-   25. Mi, W. et al., J. Immunol. 181:7550-7561 (2008).-   26. Rawool, D. B. et al., J. Immunol. 180, 5548-5557 (2008).-   27. Gallichan, W. S. et al., J. Immunol. 166, 3451-3457 (2001).-   28. Kwant, A., & Rosenthal, K. L., Vaccine. 22, 3098-3104 (2004).-   29. Tengvall, S. et al., J. Virol. 80, 5283-5291 (2006).-   30. Lindqvist, M. et al., J. Immunol. 182, 6435-6443 (2009).-   31. ChangLai, S. P. et al., Respiration. 66, 506-510 (1999).-   32. Lin, H., et al., Int. J. Pharm. 330, 23-31 (2007).-   33. Kodama, S. et al., Laryngoscope. 116, 331-335 (2006).-   34. Kelsall, B. L., & Rescigno, Nat. Immunol. 5, 1091-1095 (2004).-   35. Yoshida, M. et al., Immunity 20, 76-783 (2004).

The following Materials and Methods were used in Examples 5-10 below.

Mice, cells, antibodies, viruses. Six to eight week-old female inbredC57BL/6 mice were purchased from the National Cancer Institute orCharles River. FcRn knockout mice on a C57BL/6 background (19) were fromthe Jackson Laboratory. All mice were bred and maintained inHEPA-filtered caging units. Animal experiments were approved by theAnimal Care and Use Committee at the University of Maryland.

Madin-Darby canine kidney (MDCK) cells expressing rat FcRn were obtainedfrom Dr. Pamela Bjorkman at the California Institute of Technology. Veroand Chinese hamster ovary (CHO-K) cells were purchased from the AmericanTissue Culture Collection (ATCC). MDCK, Vero, and CHO cells weremaintained in DMEM complete medium (Invitrogen Life Technologies)supplemented with 10 mM HEPES, 10% fetal bovine serum, 2 mM L-glutamine,nonessential amino acids, and penicillin (0.1 ug/ml)/streptomycin (0.292ug/ml). Recombinant MDCK and CHO cells were also grown under 400 ug/mlof G418 if necessary. Cells from spleen or bone marrow were grown incomplete RPMI 1640 medium. All cells were maintained in a humidifiedatmosphere of 5% CO₂ at 37° C. Affinity-purified antibody for mouse FcRnwas produced as previously described (20). Purified mouse IgG andchicken IgY were from Rockland Laboratories (Gilbertsville, Pa.).HRP-conjugated donkey anti-rabbit or rabbit anti-mouse antibody waspurchased from Pierce (Rockland, Ill.); HRP-conjugated goat anti-mouseIgG1, IgG2a and IgG3 were from Southern Biotech. All DNA modifyingenzymes were purchased from New England Biolab (Ipswich, Mass.). Thepurified recombinant HIV Gag p24 proteins were from Meridian LifeScience (Cincinnati, Ohio). Mouse anti-Gag p24 hybridoma and recombinantvaccinia virus expressing Gag (rVV-Gag) were acquired from the NIH AIDSResearch & Reference Reagent Program. The rVV-Gag stocks were preparedby infection of Vero cell monolayer at a multiplicity of infection (MOI)of 0.1. DMEM complete medium was added and infected cells were culturedfor 2-3 days until the CPE appears. Viruses were released from cells bythree times freeze and thaw, cell debris was centrifuged, andsupernatant was stored at −80° C. Virus was titrated with Vero cellswith a standard plaque assay.

Western blot and SDS-PAGE gel electrophoresis. Purified proteins or celllysates were resolved on 12% SDS-PAGE gels under a reducing ornon-reducing condition. Proteins were transferred to a nitrocellulosemembranes (Schleicher & Schuell); membranes were blocked with 5% non-fatmilk, probed separately with primary antibodies for 1 hr, followed byincubation with HRP-conjugated rabbit anti-mouse or donkey anti-rabbitAb for 1 hr. All blocking, incubation, and washing steps were performedin PBST solution (PBS and 0.05% Tween 20). Proteins were visualized bythe ECL method (Pierce, Rockland, Ill.).Expression of Gag-Fc Fusion Proteins. The cDNA encoding Gag P24 from theHIV-1 isolate BH10 was amplified by PCR from a plasmid pBKBH10S providedby NIH AIDS Reference Reagent Program using forward primer:5′-ctggtcgcttccgtgctacctagaactttaaatgcatg-3′, SEQ ID NO:10 and reverseprimer: 5′-agatcccgagccacctcctccggacccacccccgcctgatcccaaaactcttgccttatg-3′, SEQ ID NO:11. The antisense primer introduces an extensionwith 12 codons for glycine and serine residues (GSSGGGSSGGSSS, SEQ IDNO:1). The Fc-fragment of mouse IgG2a containing hinge, CH2 and CH3domains, was amplified by RT-PCR from the OKT3 hybridoma. Similarly, theforward primer for IgG2a Fc has complementary glycine and serine codonsfor Gag. A mutant Fc (HQ310 and HN433) was made by oligonucleotidesite-directed mutagenesis (Clontech, Mountain View, Calif.) anddesignated as an Fc/mut as described previously (15). To construct anonlytic Fc fragment, oligonucleotide site-directed mutagenesis was usedto replace the C1q binding motif Glu318, Lys320, Lys322 with Alaresidues (21). Fusions were then performed in a PCR-based gene assemblyapproach by mixing the cDNA for Gag and the Fc fragments. All these DNAfragments were digested by BamH I and EcoR I, then ligated into anengineered pcDNA3 vector carrying a CD5 protein secretion signalsequence. Each construct was verified by DNA sequencing.

Plasmids containing the chimeric Gag-Fc/wt (SEQ ID NO:12) or Gag-Fc/mutfragment were transfected into CHO cells by Effectene (Qiagen, Valencia,Calif.). G418-resistant clones were selected for secretion of Gag-Fcfusion proteins. SDS-PAGE and Western blot were performed to assess therecombinant fusion proteins in serum-free medium (Invitrogen, Carlsbad,Calif.) using HRP conjugated rabbit anti-mouse IgG or anti-Gag p24antibody. Recombinant proteins were made from CHO cell supernatantscleaned first by ultrafiltration and purified further by affinitychromatography using Protein A Sepharose 4 Fast Flow (AmershamPharmacia, Piscataway, N.J.) for Gag-Fc/wt or goat anti-mouse IgGaffinity column (Rockland) for Gag-Fc/mut proteins. Proteinconcentrations were measured with Bradford protein assay kits (Pierce)using mouse IgG2a as standards.

In vitro and in vivo transcytosis. The in vitro IgG transport assay wasperformed as a modification of previously-described methods (22, 23).MDCK cells expressing rat FcRn (23) were grown on transwell filterinserts (Corning, Lowell, Mass.) to form a monolayer exhibitingtransepithelial electrical resistances (TER, 300 Ω·cm²) measured withplanar electrodes (World Precision Instruments). Monolayers wereequilibrated in serum free medium for 3 hr. Fusion proteins at a finalconcentration of 0.1 mg/ml were applied to the apical compartment, andincubated with DMEM medium supplied with or without 1 mg/ml of chickenIgY as competitors for 1 hr at 37° C. Transported proteins were sampledfrom the basolateral chamber and analyzed by reducing SDS-PAGE andWestern blot-ECL. For in vivo transport, 20 ug of Gag-Fc fusion proteinsor Gag alone in 20 ul of PBS were administered intranasally (i.n.) intothe wild type or FcRn KO mice that were anethesitized with 100 ul ofavertin (40 mg/ml). 8 hr later or at indicated time points, transportedproteins in sera were determined by ELISA.Mouse immunization and virus challenge. Groups of 5 mice were immunizedintranasally with 20-30 μl of 20 ug Gag-Fc/wt, Gag-Fc/mut, or Gagproteins in combination with 20 ug CpG ODN1826(5′-TCCATGACGTTCCTGACGTT-3′, (SEQ ID NO:9) (InvivoGen, San Diego,Calif.) at weeks 0 and 2, respectively. An additional group of 5 micewas mock-immunized with PBS following the same schedule. For intranasalinoculation, 20 ul proteins or PBS were applied to each nostril of miceanesthetized with 100 ul of avertin (Sigma, 40 mg/ml). Mice were kept ontheir backs under anesthesia to allow the inoculums to be taken up.Mice were challenged with viruses by intravaginal inoculation asdescribed previously (24). Five days before each inoculation, mice weretreated subcutaneously with 2 mg of medroxyprogesterone acetate(Depo-Provera). Mice were anesthetized by avertin (40 mg/ml, Sigma) andexposed intravaginally to rVV-Gag (5×10⁷ PFU) in 30 μl of PBS. Mice werekept on their backs under the anesthesia for 1 hr. Five days afterchallenge, mice were sacrificed. Paired ovary tissues were removed andhomogenized in nylon mesh. For virus titration, serially diluted ovarysamples were inoculated into Vero cells and incubated for 45 min at 37°C. The cells were washed and DMEM containing 0.8% methcellulose and 2%FBS was overlayed on the cells. The cells were cultured for 3 days, theoverlay was removed, and the cells were fixed with 3.7% formaldehyde for1 hr, and stained with 1% crystal violet.

Clear plaques were counted to determine the virus titer in terms ofplaque forming units (pfu).

Enzyme-linked immunosorbent assay (ELISA) and ELISPOT. HIV Gagp24-specific antibodies were detected in serum, bronchial lavage andvaginal fluid. High-binding ELISA plates (Maxisorp, Nunc) were coatedwith 1 μg/ml of purified p24 protein in PBS and incubated overnight at4° C. Plates were then washed three times with 0.2% Tween 20 in 50 mMTris buffer and blocked with 1% BSA in PBS for 2 hr at room temperature.All samples were diluted 10-fold in PBS then transferred to countedplates and incubated for 2 hr at room temperature. HRP-conjugated rabbitanti-mouse IgG antibody (1:2,000, Pharmingen) or anti-mousesubclass-specific antibodies (1:5000, SouthernBiotech) was added and acolorimetric assay was done with tetramethyl benzidine (KPL) and aVictor III microplate reader (Perkin Elmer). Antibody titers representthe highest dilution of samples showing a 2-fold OD₄₅₀ value overcontrols. The mean log of the end-point dilutions was determined andused to calculate the average end-point titer. Each assay was done intriplicate. Mouse cytokines IFN-γ, IL-2, and IL-4 from the cell culturesupernatant were analyzed by ELISA according to the manufacturer'sinstructions (BD Biosciences).

For measuring HIV Gag-specific antibody-producing plasma cells, 96-wellELISPOT plates (Millipore) were coated with 5 ug/ml Gag and blocked withRPMI 5% FCS (Invitrogen) for 90 min at 37° C. in 5% CO₂. Serialdilutions of bone marrow single-cell suspensions were prepared in RPMIand incubated in the coated wells for 24 hr at 37° C. in 5% CO₂. Cellswere removed; plates were washed 5 times with 0.1% Tween 20 in PBS andthen incubated with biotin labeled goat anti-mouse IgG-specific antibody(1:1500, Sigma) for 2 hr. After washing the cells with PBS, avidinconjugated HRP (1:2,000, Vector Laboratories) was added and incubatedfor 1 hr, and developed with substrate from the AEC kit (BDBiosciences). Spots were counted with an ELISPOT Reader and analyzedwith software (Zesis).

Preparation of single-cell suspensions from spleen and vaginal tissues.Spleens were made into single-cell suspensions by passing through asterile mesh screen. Cells were resuspended in Hanks' balanced saltsolution (HBSS) and counted by trypan blue dye exclusion. For eachexperiment, cells were generally pooled from 3 mice. Vaginal cells wereisolated from tissue that was excised, cut longitudinally, and mincedwith a sterile scalpel in complete RPMI 1640 culture medium. Mincedtissues (epithelium and lamina propria) were digested in complete mediumwith sterile 0.25% collagenase D. Digestion was accomplished withshaking incubation at 37° C. for 30 min. After collagenase treatment,tissues and cells were filtered through a sterile gauze mesh, washedwith RPMI 1640 medium and additional tissue debris was excluded byslow-speed centrifugation for 1 min. Cells were collected from thesupernatant by centrifugation, resuspended in HBSS and viable cells werecounted by trypan blue dye exclusion.Flow cytometry. Single cell suspensions were obtained from spleen orvaginal tissues. Erythrocytes were lysed in 0.14 M NH₄C1, 0.017 MTris-HCl at pH 7.2 on ice for 5 min. Cells were preincubated with an Fcblock (mAb to CD16-CD32, 2.4G2, PharMingen, San Diego, Calif.) andwashed in FACS buffer (HBSS, 2% bovine serum albumin, 0.01% sodiumazide). Blocked cells were incubated with specific antibody directlyconjugated to fluorsecein isothiocyante (FITC), phycoerythrin (PE),allophycocyanin (APC), cyanine dye Cy7 (Cy7), and peridinin-chlorophyllproteins (PerCP) then washed, transferred to FACS buffer, and analyzedusing a FACSAira (Becton Dickinson, Mountain View, Calif.) and FlowJosoftware (Tree Star). The mAbs (PharMingen) we used were anti-CDR,500A2; anti-CD4, RM4-5; anti-CD8, 53-6.7; anti-IFN-γ, XMG1.2; anti-B220,RA3-6B2; FAS, Jo2. Peanut agglutinin (PNA)-FITC was from Sigma. PurifiedHIV Gag proteins were labeled with Alexa Fluoro647 protein labeling kit(Invitrogen) according to the manufacturer's instruction. Cells wereincubated with isotype control antibodies to determine the backgroundfluorescence. The isotype control antibodies included in each experimentwere considered the true baseline fluorescence used to evaluate andillustrate the results for cell-specific antigen markers.T cell proliferation assay. Carboxyfluorescein diacetate succinimidylester (CFSE, 5 mM stock, Invitrogen) dilution was used to assess T cellproliferation in response to Gag antigen. CFSE was added single cellsuspensions 10⁷ cells/ml from spleens, in prewarmed PBS/0.1% BSA for a 2mM final concentration; reactions were incubated for 10 min at 37° C.then stopped with 5 volumes of ice-cold culture media and 5 min on ice.The cells were washed three times with RPMI-1640 containing 10% FCS.After labeling with CFSE, the splenic T cells were added in the presenceof HIV Gag p24 protein (20 μg/ml), medium alone, or anti-CD3 (0.1 μg/ml)and anti-CD28 (2 μg/ml) as a positive control. Cells (5×10⁵) werecultured for 4 days. The cells were then harvested and subjected to flowcytometry assay.Intracellular cytokine staining. Intracellular IFN-γ production byprimed CD4⁺ and CD8⁺ T cells was evaluated using bulk splenocytes orisolated vaginal infiltrating lymphocytes incubated for 12 hr with 20ug/ml of the purified Gag protein or medium alone. Cells were thencultured for another 6 hr in the presence of 10 μg/ml brefeldin A(Sigma) to accumulate intracellular cytokines. Cells were washed,incubated for 15 min at 4° C. with 2.4G2 mAb to block Fcγ receptors, andstained with PE-conjugated anti-mouse CD3ε and FITC conjugated anti-CD4and APC-Cy™7 conjugated anti-mouse CD8a for 30 min at 4° C. The cellswere fixed and permeabilized (Cytofix/Cytoperm Plus, BD Biosciences) andstained with APC-anti-IFN-γ (XMG 1.2) mAbs for 30 min at 4° C. (BDBiosciences). Cells were washed three times, resuspended in FACS bufferand analyzed by flow cytometry and FlowJo software. All plots were gatedon low forward and side scatter CD3+ cells.Histological analysis. Immunohistochemical staining of mouse FcRn wasperformed using an affinity-purified rabbit anti-mouse FcRn antibody(20). Briefly, lung and trachea were excised and embedded in Tissue-TekOCT compound (Miles, Elkhart, Ind.). Thin sections were cut with acryostat, transferred onto glass slides and stored at −80° C. Beforestaining, sections were fixed in ice-cold acetone for 10 min. Afterextensive washes in PBS and blocking buffer (PBS-2% bovine serumalbumin-10% normal goat serum) for 1 h, sections were incubated withaffinity-purified rabbit anti-mouse FcRn antibody followed by 488Fluoro-conjagated goat anti rabbit IgG. Tissues were washed at leastthree times with 0.1% Tween-20 in PBS. Nuclei were then labeled withDAPI for 10 min. Coverslips were mounted on slides with ProLong™antifade kit (Molecular Probes) and examined using a Zeiss LSM 510confocal fluorescence microscopy. Images were handled in Adobe Photoshop7.0.Statistical analysis. Antibody titers, serum Gag concentration, cytokineconcentration and virus titers were assessed with unpaired two-tailed ttest. GraphPad Prism 5 was the software for statistical analyses.

Example 5

Production and transcytosis of HIV Gag-Fc fusion proteins. To determinewhether HIV antigens targeted to FcRn in vivo would elicit antibody andcellular immune responses, we first generated a fusion protein HIVGag-Fc/wt by cloning HIV Gag in frame with the carboxyl terminus of theheavy chain of mouse IgG2a antibody (FIG. 1A). We used mouse IgG2a Fcfragment since mouse IgG2a, but not IgG1, is capable of binding mouseFcγRI. We also generated a Gag-Fc mutant version that does not bind FcRnby creating point mutations (HQ310 and HN431) known to prevent FcRnbinding to the Fc-domain. These same Fc mutations in IgG1 Fc are knownto exhibit a 100-fold reduction in binding to FcRn (15). In allconstructs, constant regions of the mouse IgG2a were also modified toremove the complement C1q-binding motif (21) and produce nonlytic fusionproteins. The fusion proteins were synthesized in CHO cells transfectedwith the Gag-Fc constructs. Secreted Gag-Fc fusion proteins formedmonomers under reducing conditions, but were disulfide-linked homodimersunder non-reducing conditions in Western blotting, using both theaffinity-purified anti-Gag (FIG. 6B, top panel) and anti-mouse IgG Fcantibodies (FIG. 6B, bottom panel). Functional testing of the Fc-domainwas confirmed by precipitating Gag-Fc/wt, but not for Gag-Fc/mutproteins with Staphylococcal protein A on beads. It has been shown thatprotein A and FcRn recognize overlapping amino acids of IgG Fc andmutations in this region can affect both properties. As a result,protein A effectively and competitively inhibits IgG binding to FcRn.This implies that Fc portions of IgG in the Gag-Fc/wt maintain allstructures necessary for binding FcRn.

To ascertain whether the Gag-Fc/wt, but not Gag-Fc/mut, fusion proteinsare transported by FcRn, we used an MDCK-FcRn cell line to transportGag-Fc fusion proteins. MDCK cells expressing rat FcRn and β2m have beenshown to specifically transport murine IgG in vitro (23). Hence,FcRn-dependent transcytosis of purified Gag-Fc/wt protein applied to theapical reservoir was transported to the basolateral reservoir (FIG. 6C,lanes 4&5), as detected by Western blot quantification. In contrast, theGag-Fc/mut (FIG. 6C, lanes 2&3) and chicken IgY (lanes 2-5) proteinsfailed to transport across the MDCK-FcRn monolayer, suggesting aspecific transport of the Gag-Fc/wt by FcRn. The transport was notinhibited by an excessive amount of chicken IgY which does not bind FcRn(FIG. 6C, lane 3).

We then addressed whether the Gag-Fc would appear in sera after i.n.inoculation. The expression of murine FcRn in the lung (25) was verifiedin epithelial cells of trachea and lungs, but not intestines, of adultmice compared with FcRn-KO mice (19) by immunofluorescence stainingusing a mouse FcRn specific antibody (FIG. 6D). To determine whether theGag-Fc appears in circulation after i.n. inoculation, 20 ug of theGag-Fc/wt, Gag-Fc/mut, or Gag proteins were administered i.n. andmeasured in the blood 8 hr later using ELISA. As shown in FIG. 6E,Gag-Fc/wt was detected readily in sera of wt mice, but not FcRn KOanimals. In addition, the Gag-Fc/mut or Gag alone proteins weretransported poorly; indicating that i.n. administered Gag-Fc/wtefficiently crossed the airway mucosal barrier. The transportedGag-Fc/wt proteins entered the circulation and persisted about 5 days,much longer than other proteins tested (FIG. 6F), although it wasdifficult to determine the half-life of other proteins because they weretransported so poorly. Taken together, we conclude that rodent FcRn cantransport the Gag-Fc/wt fusion protein across the polarized epithelialcell monolayer lining the airway tract in an FcRn-dependent manner. Inaddition, inactivation of the complement C1q binding motif failed toimpact FcRn-mediated transport of the Gag-Fc/wt fusion protein.

Example 6

Strong anti-Gag antibody and T cell responses after FcRn-targetedmucosal immunization. To test whether FcRn-dependent transport acrossmucosal surfaces generates immune responses against HIV vaccineantigens, wt mice were immunized i.n. with Gag-Fc or Gag proteins incombination with the CpG and boosted 2 weeks later. Use of Gag aloneallowed us to assess the transport efficiency of Gag-Fc/wt by FcRn invivo and to determine the magnitude of increased immune responses toGag-Fc/wt. DCs capturing antigen in a mucosal-associated lymphoidtissues or in lymph nodes primes T cells that home to the mucosa (18,26). To overcome the normal tolerogenic function of at least someimmature DCs in vivo, we included immunostimulatory DNA rich in CGmotifs (CpG), an agonist for Toll-like receptor-9 (16, 27). First, wedetermined whether antigen targeting to FcRn by the Gag-Fc proteinselicits antibodies specific to HIV Gag protein. Antibody responses amongimmunized animals, including PBS control mice, were assessed at varioustime points up to 56 days after the primary immunization, by measuringGag-specific serum IgG. As shown in FIG. 7A, significantly higher titersof IgG, mainly restricted to the IgG2a subclass (FIG. 7B), were seen inthe Gag-Fc/wt immunized mice when compared with the other groups. Theantibody response was further associated with germinal center (GC) inthe spleens of immunized mice. Spleens were removed 10 days after theboost. Splenocytes were gated on B220⁺ cells and stained for thepresence of peanut agglutinin (PNA) and Fas-positive B cells. Miceimmunized by the Gag-Fc/wt, but not by the Gag-Fc/mut or Gag aloneproteins, developed appreciable numbers (3.98% of total isotype-switchedB cells) of FAS⁺PNA⁺B220⁺ B cells, suggesting GC were formed afterimmunization (28, 29)(FIG. 2C). In addition, FcRn KO mice immunized withGag-Fc/wt failed to show comparable numbers of FAS⁺PNA⁺B220⁺ B cells.These results indicate that FcRn-targeted mucosal delivery of theGag-Fc/wt results in an effective B cell response.

An effective antibody response requires help from T cells (30). Sevendays after boosting, splenocytes were harvested from immunized mice andpulsed with purified Gag; IFN-γ producing T cells were measured by flowcytometry. We detected a significant number of IFN-γ producing CD4⁺(FIG. 7D, upper panel) and CD8⁺ (FIG. 7D, bottom panel) T cells inresponse to Gag in mice immunized with Gag-Fc/wt, but not in any ofother groups including mice immunized with the Gag-Fc/mut protein orFcRn KO mice immunized with the Gag-Fc/wt protein. In wild type miceimmunized with the Gag-Fc/wt proteins, about 1% of the CD4+ and 2% ofthe CD8+ T cells responded to Gag stimulation. Thus, targeting theGag-Fc/wt to FcRn via mucosal administration was at least 10-20 times aseffective at initiating IFN-γ-producing CD4+ and CD8+ T cell immunitycompared with Gag-Fc/mut or Gag-immunized mice. Cytokine responses weremainly IFN-γ and IL-2; little IL-4 was seen in cultures pulsed withantigen (FIG. 7). Mucosal immunization with Gag-Fc/wt proteins thereforeinduced strong CD4⁺ and CD8⁺ T cell responses, whereas the immunizationwith Gag-Fc/mut or the Gag-Fc/wt in FcRn KO mice did not. We concludedthat immunizing by targeting HIV antigens to FcRn together with CpG,produces a strong response to HIV Gag including both B and T cellimmunity.

Example 7

FcRn-targeted mucosal immunization significantly reduced viralreplication after challenge. To determine whether to FcRn-mediatedmucosal vaccine delivery could protect against viral infection at adistant mucosal site, we challenged intravaginally (ivag) the immunemice with 5×10⁷ pfu of virulent recombinant vaccinia virus (VV)expressing HIV-1 Gag (rVV-Gag) at four weeks after the boost. Ovarytissues were harvested at the peak of infection, day 5. Control mice(PBS) had the highest titers of rVV-Gag in ovaries after virus challenge(FIG. 8A). Mice immunized with Gag-Fc/mut or the FcRn KO mice immunizedwith Gag-Fc/wt, had high titers of rVV-Gag in their ovaries. In markedcontrast to these control mice, virus titers measured in ovary tissuesof wild type mice immunized with the Gag-Fc/wt proteins showedsignificantly lower levels of virus by day 5 after challenge (FIG. 8A).Furthermore, in comparison with Gag-Fc/mut immunized wild type mice orGag-Fc/wt-immunized FcRn KO mice, the uterine sizes were much smaller inmice immunized by Gag-Fc/wt proteins after infection with rVV-Gag,presumably because of reduced edema, hemorrhage and inflammation (FIG.8B). Control (uninfected) mice sampled at day 0 showed normal uterusmorphology. The amounts of virus detected were consistent with the grosschanges in the uteri among groups of animals. Overall these resultsdemonstrate that i.n. administration of Gag-Fc/wt proteins in wild typemice efficiently induced protective immunity. These results suggest asignificant role for the immune responses from FcRn-dependent mucosalimmunization in the control of viral infection.

Example 8

Induction of local mucosal immune responses. Sexually transmitted HIVenters through mucosal sites and spreads rapidly to distant mucosal andsystemic lymphoid tissues. Local immune responses and protection againstvirus dissemination from mucosal tissues are important factors forvaccine development. Local mucosal immunization confers maximumprotection against mucosal challenge (16, 31). Mediastinal lymph nodes(MLN) are the sites where mucosal immune responses are initiated againstvaccine antigens that reach the lung after intranasal immunization. Welooked at changes in MLN GC after FcRn-targeted mucosal delivery of theGag-Fc/wt. As shown in FIG. 9A, intranasal immunization with Gag-Fc/wtefficiently induced an increased frequency (3.93%) of FAS⁺PNA⁺B220⁺ Bcells in the MLN compared to 0.51 to 0.98% of Fas+PNA+B220+ cells inother groups by 10 days after the boost. Therefore, FcRn-targeted HIVGag mucosal immunization induced the formation of GC in draining MLN.

Antibodies, in particular secretory IgA and IgG, represent a first lineof defense on mucosal surfaces. To assess the ability of theFcRn-targeted immunization to induce Gag-specific antibody in mucosalsecretions, the bronchial alveolar lavage (BAL) specimens were collectedtwo weeks following the boost and tested for Gag-specific IgG and IgA byELISA. Furthermore, in order to determine if the antibody responsesinduced by i.n. immunization were disseminated to remote mucosal sites,vaginal washes were collected for antibody analyses. The Gag-specificIgG were increased significantly in lung lavages by 10 days after theboost (FIG. 9B) and in vaginal washes two weeks after the boost (FIG.9C) among the Gag-Fc/wt immunized mice. Low levels of the Gag-specificIgG were detected in BAL and vaginal washings of mice immunized with theGag-Fc/mut or Gag alone. Only wild type, but not FcRn KO mice thatreceived the Gag-Fc/wt, had highest levels of Gag-specific IgGantibodies in the BAL and vaginal washings suggesting the appearance ofmucosal IgG is FcRn dependent. In contrast, we only detected a smallamount of IgA in all BAL and vaginal washings (data not shown).

To address whether FcRn targeted delivery of mucosal vaccine can induceT cell immune responses in the vaginal tissue, infiltrated vaginallymphocytes were isolated from the challenged mice and pulsed with thepurified Gag; IFN-γ specific producing T cells were measured by flowcytometry. We detected significant numbers of IFN-γ producing CD4+ (FIG.9D, upper panel) and CD8+ (FIG. 9D, bottom panel) T cells in response toGag in mice immunized with the Gag-Fc/wt in comparison with othergroups, including the mice immunized with Gag-Fc/mut or the FcRn KO miceimmunized with Gag-Fc/wt proteins. Immunization with the Gag-Fc/wtfusion protein induced strong IFN-γ-producing CD4⁺ and -CD8⁺ T cellresponses, whereas immunization with the Gag-Fc/mut or the FcRn KO miceimmunized with the Gag-Fc/wt protein did not.

Example 9

FcRn targeted mucosal immunization elicits long-term humoral and T cellimmune responses. Activated B cells, can differentiate to plasma cellsthat secrete antibodies at high rate and reside in niches in the bonemarrow and others become memory B cells that respond rapidly toantigenic restimulation and contribute to the plasma cell pool serumantibody levels over a prolonged period of time (32). To determinewhether antigen targeting to FcRn-mediated IgG transfer pathway leads tolong-lasting memory B cell immune responses, splenocytes were isolated 4months after the boost and restimulated with Gag protein. Memory B cellswere barely present after immunization with control Gag or PBS but wereincreased with Gag-Fc/wt (FIG. 10A). Differences between the Gag-Fc/wtimmunized mice versus the Gag-Fc/wt immunized FcRn KO or the Gag-Fc/mutimmunized mice were statistically significant. To determine whetherantigen targeting to FcRn also elicited plasma cells that secretedGag-specific antibodies, the number of IgG-secreting plasma cells in thebone marrow were measured by ELISPOT. High numbers of Gag-specific IgGsecreting cells were present in bone marrow of mice immunized withGag-Fc/wt compared with other groups (FIG. 10B). To show whetherincreased memory B cells and antibody-secreting plasma cells correspondto a rise in IgG production and maintenance, IgG antibody in the serawere measured four months (the longest point we tested) after the boost.High titers of Gag-specific IgG antibodies were maintained in miceimmunized with the Gag-Fc/wt, but not the Gag-Fc/mut or Gag alone (FIG.10C). Immunization with the Gag-Fc/wt was about 20-fold more effective,respectively, than immunization with Gag-Fc/mut or HIV Gag alone,indicating that Gag-specific antibody persisted much longer after FcRntargeted mucosal immunization.

An important feature of memory T cells is their proliferative responseupon antigen restimulation. To test if memory T cells could be detected4 months following FcRn-targeted mucosal immunization, we measured CD4⁺and CD8⁺ T cell proliferation in response to HIV Gag antigenrestimulation. Splenocytes isolated four months after the boost werestimulated in vitro with HIV Gag (FIG. 10D). After 4 days incubation,the CFSE profiles were read on CD4- or CD8-gated T cells andsubsequently analyzed by flow cytometry. We detected significant CD4⁺(FIG. 10D, upper panel) and CD8⁺ (FIG. 10D, bottom panel) memory T cellproliferation in response to Gag restimulation, in mice immunized withGag-Fc/wt but not in other groups. The Gag-specific T cell response toGag-Fc/wt in wild type mice included a substantial memory component.Recall IL-2 and IFN-γ cytokine responses were also detected within 12-48hr of Gag restimulation among mice immunized with the Gag-Fc/wt, but notwith other groups (data not shown). Collectively, these results showthat mucosal immunization by antigen targeting to FcRn was effective ineliciting long term memory T cell immune responses to HIV Gag antigen.

To test whether memory immune responses elicited from FcRn targetedmucosal immunization are functional for resisting virus, we challengedthe immunized mice intravaginally with rVV-Gag (5×10⁷ pfu) at fourmonths after the boost. Ovary tissues were harvested 5 days afterchallenge and virus titers were measured. Mice immunized by theGag-Fc/mut, Gag alone, or FcRn KO mice immunized by Gag-Fc/wt failed tocontrol viral replication (FIG. 10E). In contrast, virus titers in ovarytissues from wild type mice immunized by Gag-Fc/wt were significantlylower at 5 days after challenge (FIG. 10E). Virus titer wassignificantly higher in either Gag-Fc/mut immunized wt mice orGag-Fc/wt-immunized FcRn KO mice, demonstrating that the protection wasfrom HIV Gag specific memory immune responses.

Discussion

A chimeric fusion protein comprised of HIV Gag protein and a modifiedmurine Fc portion from IgG, was transported efficiently across mucosalepithelium. Transported Gag-Fc persisted much longer in the blood, whichis consistent with fact that FcRn protects IgG from degradation (10).When Gag-Fc fusion protein was used for intranasal immunization, micedeveloped strong T cell (CD4+ and CD8+) and B cell responses includingpersistent memory. By introducing genetically modified Fc fragments intothe HIV Gag protein or using FcRn KO mice, we show that the capacity fortransepithelial transport and for eliciting strong immune responses bothdepended on the intact Fc sequence in the fusion protein and FcRnexpression on murine cells. Finally, immune responses elicited byintranasal immunization were sufficiently potent to protect mice frominfection at a remote mucosal site. The properties of antigen transport,antigen persistence in blood, B and T cell immune responses andprotection from virus challenge all required intact Fc sequences in thefusion protein and FcRn expression in the mouse.

FcRn-targeted immunization induced strong antibody and cellular immuneresponses against HIV Gag. Strong responses were detected at mucosal andsystemic sites. The Gag-Fc/wt fusion proteins induced strongIFN-g-producing CD8⁺ and CD4⁺ T cell responses relative to theGag-Fc/mut or HIV Gag proteins. The mucosal immunization of FcRn KO micedemonstrated that FcRn was absolutely essential for mucosalimmunization. Gag antigen targeted to FcRn increased the efficiency withwhich HIV Gag antigens engendered strong T cell immunity, when giventogether with CpG stimuli to promote DC maturation.

We also noticed that the FcRn-targeted mucosal subunit vaccine wasexceptional in inducing high levels of serum IgG production with apreference for IgG2a as the major isotype. This is not surprisingbecause the type 1 cytokine IFN-γ is associated with production ofIgG2a, whereas the type 2 cytokine IL-4 helps switching to IgG1. T cellsanalyzed in this study secreted a lesser amount of IL-4. However, we didnot distinguish between the effect of mucosal targeting or CpG in ourstudy.

An effective vaccine to block sexual transmission of HIV must be capableof eliciting protective mucosal immune responses to stall initial virusreplication, slow CD4+ depletion and inhibit rapid dissemination ofvirus from the mucosa into systemic lymphoid tissues (33). Our strategyof FcRn-targeted mucosal delivery for HIV Gag antigen, engendered strongmucosal immune responses. We observed IgG in lung and vaginal washingsand cytokine-producing T cells in vaginal tissues of immunized mice. Ofnote is the observation that levels of Gag-binding IgG levels in BAL- orvaginal washes were much higher than the specific IgA detected. Indeed,IgG is a major isotype of immunoglobulins in the lower respiratory andreproductive tracts (34, 35). Thus, IgG antibodies detected in BAL andvaginal washings may be produced locally or come from the circulation,but it was clear that HIV antigen targeted to FcRn plus adjuvantproduced strong humoral and T cell mucosal immune responses.

Perhaps our most compelling finding was that FcRn-targeted intranasalimmunization protected against intravaginal virus challenge. Viralreplication in ovary tissues was reduced in immune mice and there wasless evidence of gross pathology in the uterus. Intranasal immunizationtargets cells in the nasal lymphoid tissue (NALT) and its draining lymphnodes. To separate vaginal immune responses, antibody secreting cellsand IFN-γ producing T cells likely migrate from the airway to thegenital tract (36, 37). We used this system to test whether humoral andT cell immune responses elicited by FcRn-targeted, HIV Gag intranasaldelivery protected the distant vaginal mucosa. We know that protectiveresponses depended on intact Fc and FcRn expression and both T and Bcell responses were detected. Several immune mechanisms may account forprotection. T lymphocytes were present in the vaginal epithelium ofrVV-Gag infected mice at times coinciding with virus clearance. IFN-γproducing CD4+ and CD8+ T cells were also present in vaginal tissues ofimmune mice. IFN-γ is indispensable for resistance to genital mucosalinfections (24, 38, 39) and these strong T cell responses may alsopromote direct lysis of MHC class I or II-bearing infected cells. Theeffector/memory CD4⁺ and CD8⁺ T cells in mucosal effector sites (laminapropria) are crucial for containing initial HIV replication andsubsequent virus discrimination. Consistent with these data, previousstudies reported that the breadth of Gag specific T cell responsescorrelated with control of viral load in HIV-1-infected humans (40) andSIV (simian immunodeficiency virus)-infected rhesus macaques (41). Itmay be argued that local T cell responses observed after challenge withrVV-Gag, were due to nonspecific inflammatory responses against thechallenge virus. However, the responses were Gag specific and occurredonly in wild type mice immunization with Gag-Fc/wt. It is important tonote that antigens used in this study contained only a single HIV Gagantigen and did not include a homologous Env gp120 antigen. The observedprotection was likely provided by Gag-specific cellular immuneresponses, since it is unlikely that Gag-specific antibodies offeredsubstantial protection in blocking viral attachment and penetration intotarget cells. This may explain why Gag-Fc/wt immunized mice failed toclear virus completely after infection however, we cannot exclude aprotective role for antibody-dependent cell-mediated cytotoxicity (42).Significant amounts of Gag-specific IgG antibody were present in BAL andvaginal secretions in our study and IgG is a major protective antibodyin vaginal secretions after immunization (34). The potential roles forantibody produced from FcRn-targeted HIV gp120 antigen immunization maybe more important for full protection in humans. Additional studiesconducted to test this idea are ongoing.

FcRn-targeted mucosal immunization produced durable memory immuneresponses. Immunological memory is exemplified by increased levels ofeffector T and B cells and, functionally, by the ability to respondfaster and more vigorously to a second encounter with the vaccineantigens (43). Hence, another criterion for successful HIV vaccines isthe ability to generate durable memory responses that maintain strongimmune surveillance over lengthy intervals. These effector memoryresponses might improve vaccine efficacy by impairing viral replicationat its earliest stage or at viral entry sites (44). An obstacle for thesuccessful implementation of HIV mucosal vaccine is the production andmaintenance of a pool of memory lymphocytes. It has been difficult toimplement an HIV subunit vaccine strategy via mucosal surfaces becausethese approaches have resulted in inefficient immune responses whichwaned rapidly. However, the most striking finding in this study is thatthe FcRn targeted mucosal delivery of HIV antigen sustained high levelsof HIV Gag-specific IgG-secreting plasma cells and memory B and T cells.Presence of Gag-specific memory B cells in the spleen and long-livedplasma cells in the bone marrow may explain the high levels of IgGantibody in sera. T cell memory was also long-lived in our model. Thereason for generating potent memory T cell activity is not completelyclear, although IL-2-producing T cells were generated and may beimportant for supporting long-lived memory T cells (45).

The FcRn might contribute to increased immunity in two ways: byefficiently transporting Gag-Fc and protecting it from degradation. Itis generally believed that slow release of vaccine antigen over aprolonged time can facilitate long-term memory immune responses. As aresult, long-term retention of Gag-specific plasma cells and memorylymphocytes might be important for resistance to HIV replication andtransmission. Indeed, this conclusion was strongly supported by theobservation that protective immune responses were still present at 4months after immunization (FIG. 10E). FcRn is expressed in both theupper and central airways in non-human primates as well as in humans.Additionally, FcRn can mediate a pulmonary delivery of erythropoietin Fcfusion protein in non-human primates (Bitoni et al., 2004, PNAS 101,9763-8). Therefore, it will be of interest to determine whetherFcRn-targeted mucosal immunization is capable of eliciting the long-termprotective memory immune responses in modulation of replication andtransmission of SIV in a rhesus macaque model.

In conclusion, our study demonstrates clearly that a subunit vaccinebased on an HIV Gag-Fc fusion protein, targets the antigen to FcRn andinduces long-term immune memory protection against mucosal viruschallenge. Robust durable immune responses with protection against viruschallenge document the potential for this approach in developingvaccines against mucosal HIV exposure. This conclusion is supportedfurther by our earlier work that wild-type, but not FcRn knockout miceimmunized intranasally with genital herpesviral glycoprotein gD-Fc,resulted in complete protection when animals were challengedintravaginally with virulent HSV-2 (20). From both studies, we deducethat a FcRn-targeted HIV subunit vaccine delivers soluble antigens tomucosal DCs and gives rise to long-lived T cell help for antibodyresponses (30, 46, 47).

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What is claimed is:
 1. A vaccine comprising fusion protein comprising anFc fragment comprising the hinge region, a CH2 domain and a CH3 domainof an immunoglobulin wherein C1q motif has been mutated such that itrenders the fragment non-lytic, wherein said Fc fragment is fused at theamino-terminal to a desired antigen, wherein said antigen is Herpessimplex virus type-2 gD protein.
 2. The vaccine of claim 1, wherein saidvaccine protects against infection at a mucosal site distant anddifferent from the site of vaccine administration.
 3. The vaccine ofclaim 1, wherein said vaccine is capable of promoting and sustainingantigen specific plasma cells in a subject for at least 6 months aftervaccination.
 4. The vaccine of claim 1, wherein said mucosal epitheliumis chosen from the group consisting of lungs, intestines, colon, nasaltissue, vaginal tissue.
 5. The vaccine of claim 1, wherein said subjectis animal or human.
 6. The vaccine of claim 1, further comprising anadjuvant.
 7. The vaccine of claim 6, wherein said adjuvant is CpG.
 8. Acomposition comprising a complex comprising the vaccine of claim 1 boundto FcRn.
 9. A composition comprising a fusion protein comprising an Fcfragment, said Fc fragment comprising the hinge region, a CH2 domainwherein C1q motif has been mutated such that it renders the fragmentnon-lytic, and a CH3 domain of an immunoglobulin and wherein said Fcfragment is fused at the amino-terminal end to an antigen of interest,wherein said antigen is a pathogenic antigen or allergen.
 10. Thecomposition of claim 9, wherein said pathogenic antigen is chosen fromthe group consisting of viruses, bacteria, parasites, or fungi.
 11. Thecomposition of claim 10, wherein said virus antigen is chosen from thegroup consisting of Herpes simplex virus type 2 gD protein, HIVGag(p24), HIV gp120, influenza HA, influenza NA, influenza M2,respiratory syncytia virus F protein, respiratory syncytia virus Gprotein, and human papilloma virus protein.
 12. The composition of claim10, wherein said bacterial antigen is chosen from the group consistingof mycobacterium tuberculosis Ag85B, mycobacterium tuberculosis ESAT6,Streptococcus pneumonia PspA, Streptococcus pneumonia PsaA, andStreptococcus pneumonia CPbA.
 13. An immunogenic composition comprisingthe composition of claim
 9. 14. The immunogenic composition of claim 13further comprising an adjuvant.
 15. The immunogenic composition of claim14 wherein said adjuvant is CpG.
 16. A composition comprising a fusionprotein comprising the hinge region, a CH2 domain wherein C1q motif hasbeen mutated such that it renders the fragment non-lytic, a CH3 domainand excluding a CH1 domain of an immunoglobulin, wherein said Fcfragment is fused at the amino-terminal end to an antigen, wherein saidantigen is a pathogenic antigen or an allergen.
 17. A method forstimulating a mucosal T cell immune response in a subject comprisingadministering an effective amount of the vaccine of claim
 1. 18. Themethod of claim 17 further comprising administering an adjuvant withsaid vaccine.
 19. The method of claim 18 wherein said adjuvant is CpG.20. A method for transporting the vaccine according to claim 1 across amucosal barrier said method comprising administering said vaccine to amucosal epithelia.
 21. The method of claim 20 wherein said mucosalbarrier is the respiratory mucosal barrier and said administration isintranasal.
 22. The method of claim 20 further comprising administeringan adjuvant with said fusion protein.
 23. The method of claim 21 whereinsaid adjuvant is CpG.
 24. A method for inducing an antibody and cellularimmune response at a mucosal and systemic site against an antigen ofinterest wherein said antigen is HSV-2 gD, said method comprisingadministering the vaccine of claim 1 to a mucosal epithelium.
 25. Themethod of claim 24 further comprising administering an adjuvant withsaid vaccine.
 26. The method of claim 25 wherein said adjuvant is CpG.27. A method for stimulating a mucosal T cell immune response in asubject comprising administering an effective amount of a fusion proteincomprising Fc fragment consisting essentially of the hinge region, a CH2domain and a CH3 domain of an immunoglobulin wherein C1q motif has beenmutated such that it renders the fragment non-lytic, fused at theamino-terminal to an antigen of interest, wherein said antigen is apathogenic antigen or allergen, said administration being to a mucosalepithelium.
 28. The method of claim 27 further comprising administeringan adjuvant with said fusion protein.
 29. The method of claim 28 whereinsaid adjuvant is CpG.
 30. A method for mucosal delivery of an antigen,wherein said antigen is a pathogenic antigen or an allergen, said methodcomprising fusing said antigen to Fc fragment comprising the hingeregion, a CH2 domain wherein C1q motif has been mutated such that itrenders the fragment non-lytic, and a CH3 domain of an immunoglobulin,wherein said Fc fragment is fused at the amino-terminal end to saidantigen to create a fusion protein, and administering said fusionprotein composition to a mucosal epithelium.
 31. The method of claim 30wherein said antigen is HSV-2 gD.
 32. The method of claim 30 whereinsaid pathogenic antigen is chosen from the group consisting of viruses,bacteria, parasites, or fungi.
 33. The method of claim 32 wherein saidvirus antigen is chosen from the group consisting of herpes simplexvirus type 2 gD protein, HIV Gag(p24), HIV gp120, influenza HA,influenza NA, influenza M2, respiratory syncytia virus F protein,respiratory syncytia virus G protein, and human papilloma virus protein.34. The method of claim 32 wherein said bacterial antigen is chosen fromthe group consisting of mycobacterium tuberculosis Ag85B, mycobacteriumtuberculosis ESAT6, Streptococcus pneumonia PspA, Streptococcuspneumonia PsaA, and Streptococcus pneumonia CPbA.